JP2010528582A - RNase H2 complex and gene thereof - Google Patents

Abstract

Provided are genes related to RNase II2, ie, RNASEH2A, RNASEH2B and RNASEH2C, as well as proteins encoded by the genes. Recombinant polynucleotides containing these genes are sometimes described as vectors. Recombinant RNase H2 is also described along with assays that assess the effect of the test substance or modified (mutated) RNase H2 on the activity of the components.

Description

  The present invention relates to genes encoding components of ribonuclease (RNase) H2, recombinant RNase H2, and agonists, antagonists and modulators useful in manipulating the immune response for the purpose of treating viral infections, autoimmune diseases, etc. Relates to an assay for identifying.

  RNase H is an enzyme complex that performs endonucleolytic cleavage of ribonucleotides in RNA / DNA complexes. RNase H is frequently used in molecular biology and is used to perform RNA template degradation after reverse transcription of cDNA.

  RNase H has two classes (1 / I and 2 / II) with different biochemical characteristics. RNase H2 plays a major role in cellular RNase H activity in both human and yeast. RNase H2 is thought to be involved in the removal of RNA primers from Okazaki fragments on the lagging strand in DNA replication and the excision of single ribonucleotides in DNA-RNA duplexes. In S. cerevisiae, Rnh2Bp and Rnh2Cp can be co-purified with the catalytic subunit Rnh2Ap and both can fully reconstitute RNase H2 activity. In humans, RNASEH2A, the catalytic subunit of RNase H2, has been identified by biochemical purification methods and has clear sequence homology with yeast orthologs. However, another human protein, AYP1 (now called RNASEH2C), which was co-purified with RNASEH2A protein (Frank et al., PNAS USA 95: 12872-7, 1998), except for yeast, the Rnh2Bp and Rnh2Cp subunits No orthologs have been identified.

  Icardi Gaucher syndrome (AGS) is an autosomal inherited disease of unknown etiology. AGS clinically manifests as severe neuropathy, progressive microcephaly, convulsions, dystonia posture and psychomotor developmental delay. In children, death often occurs (Goutieres, Brain Dev 27: 201-206, 2005). AGS shows strong trait similarity to congenital brain virus infection (Aicardi & Goutieres, Ann Neurol 15: 49-54, 1984), and the increased IFN-α levels seen in AGS are also hosted by viral infection. Is similar to the immune response shown by Therefore, there is no consensus on the etiology of AGS in the literature. In particular, opinions differ on whether AGS is due to genetic susceptibility to viral pathogens or due to mutations in the regulatory function of the immune response in the host.

  Crow et al. (Am J Hum Genet 67: 213-221, 2000 and J Med Genet 4O: 183-187, 2003) identified two AGS loci, which are AGS1 on chromosome 3p21 and 13q14.3 on chromosome 13q14.3. (Ali et al., J Med Genet 43 (5): 444-50, May 2006. Epub 20th May 2005).

  The inventors have discovered that AGS2 is a component of the human RNase H2 complex and has fully identified this gene. AGS2 showed a ubiquitous expression pattern, and there was no human paralog that could be predicted by predicting domain structure and function. The remote orthologue cerevisiae (amino acid sequence homology <5%). The inventors have also identified the AGS3 and AGS4 genes. The proteins expressed from these three types of AGS genes together constitute part of the RNase H2 complex.

  AGS is highly phenotypically similar to viral infections, but this similarity is not limited to the brain. Some cases may have symptoms other than neurological features (thrombocytopenia, hepatosplenomegaly, increased liver transaminase levels), and are common with other genetic diseases that have similar symptoms to viral infections It is suggested that there is a part.

  Similarities between AGS and systemic lupus erythematosus (SLE) have also been noted (see Alarcan-Riquelme, Nat Genet 38: 866-867, 27th July 2006). In particular, SLE presents vasculitis-type skin lesions at the tip, and in this case, immunoglobulin deposition is observed at the dermal-epidermal junction (see Crow et al., Nat Genet 38: 917-920, 2006). . In a recently reported case of familial cutaneous lupus erythematosus with the same skin lesion, a common change in the AGS1 gene was observed (Lee-Kirsch et al., Am J Hum Genet 79: 731-737, 19 July 2006). In AGS, hypergammaglobulinemia and autoantibodies have been reported (see Crow et al., Nat Genet 38: 917-920, 2006). Furthermore, in SLE, elevated levels of CSF interferon alpha have been observed in lupus encephalitis and systemic. Intracranial basal ganglia calcification also occurs in about 30% of patients with central nervous system SLE.

  As a result of observations by the inventors, it was first thought that AGS, SLE and viral infections may have a common etiology related to RNase H2.

  Importantly, the inventors have identified that the AGS4 gene has a G37S mutation in the F39 family. In this case, the patient exhibits both pseudo-TORCH syndrome and AGS pathology, which has been shown to have a common molecular cause. (Pseudo-TORCH syndrome is a genetic disease in which children present symptoms similar to toxoplasmosis, rubella, CMV and HSV infections.) Therefore, pathological traits resulting from mutations in the RNase H2 complex are: It is believed that it is much broader than conventional AGS, and the genetic causes present in many cases with “congenital viral infections” may not be currently recognized.

Therefore, the present invention provides a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3, or 5 or a homologue thereof. SEQ ID NO: 1 shows the base sequence of AGS2 (RNASEH2B), SEQ ID NO: 3 shows the base sequence of AGS3 (RNASEH2C), and SEQ IN No: 5 shows the base sequence of AGS4 (RNASEH2A).
These polynucleotide sequences are NM_024570 (AGS2 / RNASEH2B, formerly named FLJ11712), AF312034 (AGS3 / RNASEH2C, formerly named AYP1), NM_006397 (AGS4 / AGS4 / AGS4 / NCSE). RNASEH2A) is reported under the accession number (the function is unknown).

Neuroimaging and clinical findings in Icardi-Gaucher syndrome (a) Axial CT image showing calcification of the basal ganglia. (B) MRIT2-weighted axial image showing high signal intensity affecting white matter, particularly the frontal lobe. For comparison, (c) an axial CT image of a congenital HIV patient showing calcification of the basal ganglia (from Belman et al., Neurology 36: 11 92-1 199 (1 986)). The figure which shows the location of the specified mutation of the essential region of AGS2, and RNASEH2B (formerly called FLJ11712) gene. (A) Genetic map of chromosome 13q14.1 indicating the AGS2 locus. This locus is identified by overlaying homozygous chromosomal segments of non-family families and is located between the microsatellite markers AC137880TG19 and D13S788. (B) The physical map of the 571 kbp essential region contains four genes with annotation information (UCSC Genome Browser May 2004 assembly). (C) RNASEH2B (FLJll712) is composed of a 47 kbp base sequence containing 11 exons and encodes a protein consisting of 308 amino acids. The coding region is indicated by diagonal lines. The position of the mutation is indicated by an arrow and indicated by a relative position counted from the translation start site. Corresponding amino acid changes are shown in bold. The figure showing the AGS3 region, the RNASEH2C (AYP1) gene, its mutations and sequence conservation in other species. (A) Genetic map of chromosome 11q13.1. The essential region is identified by linkage disequilibrium data in a Pakistani family and is located between D11S4205 and D11S987. The RNASEH2C (AYP1) gene is in the range of 1.4 kbp from the position of 65.2 Mbp in the genome sequence from telomere to centromere on the minus strand. (B) The RNASEH2C (AYP1) gene contains 4 exons (the coding region is indicated by diagonal lines) and encodes a protein consisting of 164 amino acids. The position of the mutation is indicated by an arrow and indicated by a relative position counted from the translation start site. Corresponding amino acid changes are shown in bold. (C) Both mutations occur at residues that are conserved in mammals. Sequence in the vicinity of the mutation site in the region of the mammalian RNASEH2C protein. Hs, Homo sapiens (human); Bt, Bos Taurus (bovine); Cf Canis familiaris (dog); Mm, Mus musculus (house mouse); Rn, Rattus nowegicus (rat). The substituted amino acid is indicated above the sequence. (D, e) Sequencing of the RNASEH2C (AYP1) mutation site by electrophoresis. (D) c. 428A> T (e) c. 205C> T. FIG. 4 RNASEH2A gene, genomic location, genomic structure and mutation location. (A) Genetic map of chromosome 19p13.13. RNASEH2A is in a region where two child patients (family F39) born from a cousin or cousin parent have homozygous SNPs (SNPs A-1550961, 1606327, 1606325), and SNPs A-1515950 and A-1508018 Define areas that may be genetically related to (B) Gene structure of RNASEH2A. RNASEH2A exists from the 12.8 Mbp position of the genomic sequence (UCSC Genome Browser May 2004 assembly) over 7 kbp. It contains 8 exons and encodes a protein of 299 amino acids. (C) The G37S mutation occurs at residues that are conserved in a wide range from bacteria to humans. Hs, Homo sapiens (human); Bt, Bos Taurus (cow); Cf Canis familiaris (dog); Mm, Mus musculus (mouse); Rn, Rattus nowegicus (rat); Ce, Caenorhabditis elegans; Sp, Schizosaccharomyces pombe (fission yeast); Sc, Saccharomyces cerevisiae (budding yeast); Ec, Escherichia coli (E. coli); Ph, Pyrococcus horikoshii (hyperthermophile). (D) Electrophoresis result of RNASEH2A mutation site. (E) Three-dimensional structure of the RNASEH2A catalytic site estimated using the determined crystal structure of type 2 RNase H protein as a model. The mutated G37 residue (center) is located in the vicinity of the amino acid residue considered as the active site and substrate binding site (Chapados et al., J Mol Biol 307: 541-556 (2001)). Human RNASEH2B (FYJ111712), RNASEH2C (AYP1) and RNASEH2A, when expressed in mammalian cells, form a type II ribonuclease H complex with enzymatic activity. (A) Human RNase H2 complex and the corresponding S. Schematic diagram of cerevisiae complex. (B) FYJ11712 and AYP1 tagged with T7 epitope co-immunoprecipitate (IP) with RNASEH2A tagged with myc tag. (C, d) RNASEH2A / B / C complex exhibits ribonuclease H activity. (C) Enzyme activity was examined by heteroduplex of three kinds of oligonucleotides. Oligo C is a hybrid of a 3′-end fluorescently tagged RNA oligonucleotide and a complementary 5′-end DABCYL-labeled DNA oligonucleotide, and is a substrate that can be degraded by any ribonuclease H (DABCYL is an enzyme). Fluorescence is quenched until resolved from the fluorescently labeled 3 ′ end fragment by Oligo B is a substrate capable of degrading only type II ribonuclease H. This is a DNA duplex in which the 15'th fluorescently labeled oligonucleotide strand is replaced with one ribonucleotide. Oligo A is an RNA: DNA hybrid similar to oligo C, but is not subject to enzymatic degradation. This is because the 3'-end fluorescently labeled oligonucleotide is synthesized with 2'O-methyl RNA nucleotides. (D) Type II ribonuclease H activity can be measured by immunoprecipitation using a HEK293T cell extract containing RNASEH2A / B / C with an epitope tag. Myc IP was measured with a mouse anti-myc antibody, and IgG IP was performed using normal mouse IgG immunoglobulin as a control for immunoprecipitation. A vector refers to a cell that has been transduced with the pCGT-Dest and pcDNA3.1mychis vectors. Error bars indicate standard error (SEM). Mutations in RNASEH2A reduce RNase H activity. (A) Immunoprecipitation result of RNase H2 complex extracted from HEK293T cells in which RNASEH2B and RNASEH2C with a wild-type tag were combined and co-introduced with an RNASEH2A protein mutant. It is shown that the G37S mutation in RNASEH2A does not interfere with complex formation. In represents “input”, IP represents “immunoprecipitation”, and WT represents “wild type”. (B) Mutations in RNASEH2A reduce enzyme activity. Fluorescent RNase H activity assay on the same sample as the immunoprecipitation complex shown in (a). Error bars indicate standard error (SEM). The AGS2 essential section is limited by microsatellite genotyping in two non-family families. The homozygous junction marker region is surrounded by a line. Representative eukaryotic (a) RNASEH2B / Rnh2Bp homolog sequence. Amino acids substituted in AGS patients are indicated by white letters on a black background, and substituted amino acids are indicated above them. Human AYP1 and S. cerevisiae. The amino acid sequence of Kluyveromyces walii used for examining the homology with cerevisiae Rnh2Cp is shown in (b). The alignment was performed using CHROMA software (Goodstadt et al., Bioinformatics 17: 845-846 (2001)), and the percentage of consensus sequences in eukaryotes was 80%. Residues excluded from the alignment are shown in parentheses. The hyphen (-) is used for sequence display purposes and does not indicate a missing amino acid. The GI number is shown to the right of the sequence. The symbols of the consensus sequence are as follows. a: aromatic (FHWY), b: large (EFHIKLMQRWY), c: charged (DEHKR), h: hydrophobic (ACFGHILMTVWY), l: aliphatic (ILV), p: polar (CDEHKNQRST), s: small ( ACDGNPSTV), Ser / Thr (ST), +: positively charged (HKR),-: negatively charged (DE). The abbreviations of the species are as follows. Ag: Anopheles gambiae, Am: Apis mellifera, An: Aspergillus nidulans, Bt: Bos taurus, Ca: Candida albicans, Ce: Caenorhabditis elegans, Cf: Canis familiaris, Cg: Candida glabrata, Cp: Cryptosporidium parvum, Dd: Dictyostelium discoideum, Dh: Debaryomyces hansenii, Dm: Drosophila melanogaster, Dr: Danio rerio, Eg: Eremotherium gossypii, Gz: Gibberella zeae, Hs: hom iens, KI: Kluyveromyces lactis, Kw: Kluyveromyces waltii, Mm: Mus musculus, Nc: Neurospora crassa, Os: Oryza sativa, Rn: Rattus norvegicus, Sb: Saccharomyces bayanus, Sca: Saccharomyces castellii, Sce: Saccharomyces cerevisiae, Skl: Saccharomyces kluyveri, Sku: Saccharomyces kudriavzevii, Sm: Saccharomyces mikatae, Spa: Saccharomyces paradoxus, Spo: Schizosaccha omyces pombe, Tb: Trypanosoma brucei, Tc: Trypanosoma cruzi, Tn: Tetraodon nigroviridis, Xt: Xenopus tropicalis and YI: Yarrowia lipolytica. Representative eukaryotic (b) RNASEH2C / Rnh2Cp homolog sequence. Others are the same as FIG. 8A. AGS3 gene map of chromosome 11q13.2. Microsatellite genotyping results in 6 Asian families. What is considered an ancestral haplotype is shown in bold, and the homozygous marker region is surrounded by a line. RNase H activity assay of polyinosine-polycytosine (poly (I: C)) treated HCT116 cells. A: enzyme-resistant oligonucleotide substrate, B: RNase H2-specific substrate, C: ribonuclease H substrate. Western blotting of HeK293 cell lysates with affinity purified polyclonal antibodies against sheep-derived anti-RNase H2A / B / C complexes. Here, RNase H2A / B. The C complex protein has been overexpressed with an epitope-tagged vector. The antibody allows detection of overexpressed tagged mammalian RNases H2A, H2B and H2C (two lines visible below). * A band that seems to be due to endogenous substances. Schematic diagram of RNASEH2B ^A177T targeting construct introduced into ES cells. The targeting construct is made by inserting a neomycin resistance gene cassette sandwiched between LoxP sequences (indicated by triangles) between exons 6 and 7, in addition to the 2.5 kb arm of the homologous sequence derived from the RNase H2B locus. . The introduction of a nucleotide change (indicated by an arrow) results in an alanine to threonine mutation at codon 177 that is common in AGS patients. This construct has been used for homologous recombination to create an ES cell line that is currently used for blastocyst transfer to create a knock-in mouse model of AGS. RNase H2 activity kinetic assay against wild type LCL (WT LCL), RNase H2A G37S and RNase H2B A177T in immortalized lymphoblastoid cell lines of AGS patients. In cell lines that show homozygous mutations in the RNase H2 subunit, enzyme activity is weaker than in wild-type lymphoblastoid cell lines (WT). Endpoint fluorescent RNase H assay showing reduced RNase H2 enzyme activity in lymphoblastoid cell lines of AGS patients. a: SDS PAGE gel electrophoresis of soluble recombinant RNase H2 protein complex. b: Enzymatic activity by recombinant GST, wild type GST-RNase H2 complex and GST-RNase H2 complex with G37S mutation in A subunit.

  Here, the “homologue” of a polynucleotide is a modification of a polynucleotide by deletion, substitution or addition of a nucleic acid, and at least 65% or more of the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 Homology, eg, having at least 70% or, for example, at least 74% homology. In one embodiment, the polynucleotide has a homology of 75% or 80% or more, preferably 85%, to the base sequence shown in SEQ ID NO: 1, 3 or 5. “Homolog” includes an orthologous gene. This gene refers to an equivalent gene in different species.

  In one embodiment, the homolog has 90% or more homology when evaluated by direct sequencing or sequence comparison with respect to the base sequence shown in SEQ ID NO: 1, 3, or 5, for example, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

  Sequence homology can be determined by any homology search algorithm such as direct best-fit sequence alignment method or BLAST. A description of BLAST is given in Altschul et al., In J Mol Biol 25: 403 (1990). The alignment homology score S is calculated as the sum of the replacement score and the gap score. “Substantial homology” refers to a case where the expected value (E) is low when evaluated by BLAST. The expected value is an alignment having a score equal to or higher than S, and indicates the number of alignments detected by chance when a database search is performed. The lower the E value, the more significant it is.

  In particular, modifications to the base sequence that do not affect the expressed amino acid (due to redundancy of the genetic code) fall under the definition of “homolog”. The “homolog” includes a polynucleotide that can hybridize with a polynucleotide having 15 consecutive bases of any sequence of SEQ ID NO: 1, 3, or 5, preferably under stringent conditions. Applicable. In one embodiment, the polynucleotide comprises a polynucleotide having 20 or more contiguous bases (eg, 25-50 contiguous bases) of any sequence of SEQ ID NOs: 1, 3 or 5, preferably Hybridizes under stringent conditions.

Stable hybridization of polynucleic acids occurs by base pairing by hydrogen bonding. Base pairing by hydrogen bonding is affected by the degree of complementarity of the two polynucleotide strands contained in the double-stranded structure and the conditions under which hybridization occurs. In particular, salt concentration and temperature affect hybridization. One skilled in the art will appreciate that the effective melting temperature (E Tm) of a polynucleotide duplex is given by the following equation:
ETm = 81.5 + 16.6 (logM [Na ⁺ ]) +
0.41 (% G + C) -0.72 (% formamide)

  When hybridization is performed under stringent conditions, only highly complementary base pair sequences remain as duplexes. As used herein, “stringent conditions” for hybridization refers to washing with 0.1 × SSC at 60 ° C. to 68 ° C. An appropriate concentration of SDS may be arbitrarily added to the washing conditions, for example, 0.1% SDS may be added.

  The polynucleotide may be DNA or RNA, and may be single-stranded or double-stranded. Double-stranded DNA (eg, cDNA) can usually be conveniently applied in most cases. The polynucleotide may be in the form of a vector, for example an expression vector.

  The polynucleotide of the present invention may be an isolated polynucleotide or a recombinant polynucleotide. The polynucleotide can be incorporated into an expression vector or a cloning vector. Such a vector can be used for transfection and transformation of a host cell, and the host cell may be cultured according to a known method using a conventional culture medium.

  Methods for incorporating clone DNA into an appropriate vector, methods for transfection and transformation of host cells, and methods for selecting cells that have been transfected and transformed are all known to those skilled in the art, and many suitable methods are available. (See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001).

  Suitable host cells include bacterial, yeast, insect, mammalian and plant cells. Usually, a host cell that is compatible with the vector to be used is selected.

In one embodiment, the host cell may be a human or non-human ES cell.
In another aspect, the present invention provides a lymphoblastoid cell line that expresses mutant RNase H2.

  “Mutant” with respect to proteins (eg, RNase H2A, RNase H2B, RNase H2C) refers to a protein having an amino acid sequence different from the wild-type amino acid sequence. The wild type sequence of RNase H2B is shown in SEQ ID NO: 2, the wild type sequence of RNase H2C is shown in SEQ ID NO: 4, and the wild type sequence of RNase H2A is shown in SEQ ID NO: 6.

  Mutant proteins can exhibit different functions and activities than wild-type proteins, but this is not essential. “Mutant” with respect to a protein complex (eg, RNase H2) refers to a complex in which at least one of the proteins constituting the complex is a previously defined mutant protein. The mutant protein complex can exhibit different functions and activities than the wild-type complex, but this is not essential.

  In one embodiment, the present invention provides a recombinant polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or a homologue thereof and a protein encoded by the sequence.

  In one embodiment, the present invention provides a recombinant polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 or a homologue thereof and a protein encoded by the sequence.

In one embodiment, the present invention provides a recombinant polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 or a homologue thereof and a protein encoded by the sequence.
In yet another aspect, the present invention provides a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4 or 6 or a homologue thereof.

  Here, the “homologue” of a protein is a protein that is modified by deletion, substitution, or addition of amino acids, and is at least 65% homologous to the amino acid sequence shown in SEQ ID NO: 2, 4, or 6. , Eg, having at least 66% homology, eg, at least 70% homology. In one embodiment, the protein has at least 75% homology or 80% homology, preferably 85% homology to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. In one embodiment, the homolog has 90% or more homology to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. “Homolog” includes orthologous proteins from different species.

  The present invention also provides a protein encoded by any nucleotide sequence of SEQ ID NO: 1, 3, or 5 or a homologue thereof. The base sequence may be a part of a polynucleotide, and the polynucleotide may be any recombinant polynucleotide. The polynucleotide can form part of a longer polynucleotide and can further form part of a vector.

In one embodiment, the present invention provides a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a homologue thereof.
In one embodiment, the present invention provides a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or a homologue thereof.

In one embodiment, the present invention provides a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or a homologue thereof.
In one embodiment, the proteins of the invention can form part of a chimeric (fusion) protein.

  For clarity, “protein” as used herein also means a peptide or polypeptide, and does not indicate a specific size of the polymer.

In yet another aspect, the invention is a recombinant of RNase H2, comprising
i) a protein encoded by the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or a homolog thereof; and ii) a protein comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 2 or SEQ ID NO: 6 or a homolog thereof. A recombinant of RNase H2 is provided.

In one aspect, the invention is a recombinant of RNase H2, comprising
i) RNASEH2B encoded by the nucleotide sequence of SEQ ID NO: 1 or a homologue thereof, or RNASEH2B having the amino acid sequence of SEQ ID NO: 2 or a homologue thereof,
ii) RNASEH2C encoded by the nucleotide sequence of SEQ ID NO: 3 or a homolog thereof, or RNASEH2C having the amino acid sequence of SEQ ID NO: 4 or a homolog thereof,
iii) providing an RNase H2 recombinant comprising at least one of RNASEH2A encoded by the nucleotide sequence of SEQ ID NO: 5 or a homolog thereof or RNASEH2A having the amino acid sequence of SEQ ID NO: 6 or a homolog thereof To do.

  In one embodiment, at least two of components i), ii) and iii) are included (eg, i) and iii), i) and ii), or ii) and iii)). In one embodiment, all three components i), ii) and iii) are included. Other components of the composite may optionally be included.

  Recombinant RNase H2 complexes are useful in molecular biology, particularly in processes that degrade or inhibit DNA-RNA hybrids in cDNA production and other processes. It is also useful when cleaving a DNA duplex in which single or plural ribonucleotides are embedded. The recombinant RNase H2 complex is also useful in assays to identify compounds that can alter the substrate specificity and activity of the complex. A compound capable of altering the activity of a complex may be an activator (agonist) or an inactivator (antagonist) that acts on any one of the components or the entire complex. Alternatively, compounds that can alter enzyme activity may act on upstream regulators of RNase H2. The assay may be a cellular assay or a protein assay. One or more components of the complex may optionally be mutated from the wild type, eg, G37S of the AGS4 gene. The effect of such mutations on the activity of the complex and its substrate specificity can be assessed by assays.

  Also, any one of components i), ii) or iii) can be used independently of one or both of the remaining two components in molecular biology and assays.

  Since RNase H2 plays a major role in RNase H activity in cells, reducing the activity of this enzyme has a significant impact on cellular processes that depend on the metabolism of RNA-DNA hybrids. It is thought to be the cause of the pathogenesis of autoimmune diseases. Such autoimmune diseases include, but are not limited to, bacterial infections, particularly viral infections, in addition to AGS and SLE. Other bacterial infections include bacterial infections and fungal infections. With regard to viral infections, it is particularly necessary to mention pandemic viral infections. For example, there is a pandemic due to influenza virus, and the innate immune response is inappropriately inactivated, leading to high mortality.

  RNase H2 has been proposed to be involved in the function of removing the RNA primer of the Okazaki fragment during the lagging strand DNA replication process. S. cerevisiae lacking Rnh2. In the case of cerevisiae, the viability is not affected, but the sensitivity to hydroxyurea is increased.

  RNase H2, unlike type 1 RNase H, can recognize a single ribonucleotide embedded in DNA. This enzyme is therefore important in recognizing and processing ribonucleotides improperly incorporated into genomic DNA. Such misincorporation occurs frequently in the dNTP deficient state, which provides another explanation for the high sensitivity to hydroxyurea in Rnh2 mutants.

  When single-stranded RNA binds to one of the double-stranded DNAs, the other DNA strand moves by forming an “R-loop” of single-stranded DNA. Such a loop also occurs when RNA binding protein function is inhibited after transcription in eukaryotic cells. RNase H is considered to have a function of suppressing the occurrence of such a structure and the resulting instability of genomic DNA.

  Thus, inactivation of RNase H activity can have serious consequences for DNA synthesis, removal of ribonucleotides mistakenly incorporated into DNA, and suppression of R-loop formation.

  Reverse transcription is an essential process for the replication of HIV and other retroviruses and is an important drug target for antiviral therapy. The DNA-RNA hybrid formed by this process can play an important role in the antiviral action because it can be disrupted by endogenous RNase H. Thus, RNase H2 mutations may impair the host antiviral defense response and cause AGS as a result of normal viral pathogens.

Alternatively, immunodeficiency is thought to be the cause of AGS and SLE, and when the endogenous RNA-DNA hybrid level is increased due to a decrease in cellular RNase H activity, immunodeficiency May have an important effect on bacterial infections. dsRNA and dsDNA are activators of innate immunity and activate production of type I interferon (Kawai et al., Nat lmmunol 7: 131-137, 2006 and Krieg et al., Annu Rev lmmunol 20: 709 -760, 2002). It is considered that multisystem autoimmune diseases may be caused as a result of circulating nucleic acids due to damage to other nucleases and a decrease in the function of removing nucleic acids released from dead cells to the outside of cells. Evidence supporting this is that SLE patients may have heterozygous mutations in DNase 1 and that DNase 1 ^{− / −} mice exhibit an erythema-like phenotype (Napirei et al. al., Nat Genet 25: 177-181, 2000). Furthermore, it has recently been reported that DNase II ^{− / −} | fnR ^{− / −} mice exhibit chronic polyarthritis resembling rheumatoid arthritis (see Kawane et al., Nature 443: 998-1002, October 2006). It is believed that RNA-DNA hybrids stimulate innate immunity and explain why high interferon alpha levels useful for diagnosis of AGS increase. Similarly, impairment of RNase H2 function may increase the concentration of endogenous RNA-DNA hybrid, and the increase in concentration may stimulate the production of interferon α by the same mechanism as in the case of dsRNA or dsDNA. From the above, the neuropathological features of AGS can be explained by interferon α produced in excess in the central nervous system, which has been shown in a transgenic mouse model, in which glial cells Interferon-alpha is always expressed in the brain (see Campbell et al., Brain Res 835146-61, 1999).

  The relationship between SLE and autoimmune diseases has been shown in recent studies, for example, Kelly et al., Arthritis Rheum 54: 1557-1567, 2006, where dsRNA binds to TLRs to produce IFN. Lovgren et al., Arthritis Rheum 50: 1861-1872, 2004 shows that an anti-RNA Ab is required for IFN induction in an in vitro SLE assay, and Sigurdsson et al. Am J Hum. Genet 76: 528-537, 2005, SLE is caused by dysfunction of the IFN pathway, and Graham et al., Proc. Natl. Acad. Sci USA 104: 6758-6763, 2007 It shows the association between mutation and increased risk of SLE.

  Recent theories have reported that innate immune responses to influenza share a common etiology with SLE. For example, Cheung et al., Lancet 360: 1831-1837, 2002 shows that an increase in TNF-α is associated with worsening of the pathology due to H5N1 / 97, and Kobasa et al., Nature 445: 3l9-323, 2007 reported that an atypical innate immune response was seen against a 1918 influenza virus in non-human primates, Lipatov et al., Journal of General Virology 86: 1121-1130, 2005 Shows that the balance of cytokines was lost in nine models infected with H5N1 influenza.

Accordingly, providing a compound capable of altering the activity or substrate specificity of RNase H2 is beneficial for the following purposes:
a) Increase RNase H2 activity to suppress AGS symptoms.
b) Increase RNase H2 activity to reduce microbial infections. Among them, there are bacterial infections and viral infections, but it is not limited to these. For retrovirus infection (such as HIV infection), special mention is required. In this case, a DNA-RNA hybrid is formed when virus replication occurs in the host cell. In addition, it is necessary to refer to pandemic strains of influenza. For example, a virus strain with a high mortality rate such as a 1918 influenza virus strain or an H5N1 influenza virus strain may not activate the innate immune reaction sufficiently. (See Cheung et al., The Lancet 360: 1831-1837, 2002 and Lipatov et al., Journal of General Virology 86: 1121-1130, 2005).
c) Increasing RNase H2 activity to reduce genomic DNA instability, particularly to suppress R-loop formation.
d) Increase RNase H2 activity to suppress autoimmune diseases. Examples of autoimmune diseases are systemic lupus erythematosus (SLE), celiac disease, Crohn's disease, diabetes (type I), Goodpasture syndrome, Graves' disease, Addison's disease, rheumatoid arthritis, psoriasis and multiple sclerosis However, it is not limited to these.
e) Increase RNase H2 activity to increase the efficiency of molecular biological reactions by the complex, such as the production of cDNA.
f) Reduce RNase H2 activity to increase the efficiency of gene therapy with viruses. (RNase H2 activity forms part of a host response that inhibits the usefulness of this therapy. Decreasing RNase H2 activity leads to at least partial suppression of this host response.)
g) Reduce RNase H2 activity to stimulate a host immune response to viral infection (see Akwa et al., J. Immunol 161: 5016-26, 1998).

Furthermore, the present invention provides assays for identifying activators or inactivators of RNase H2 or its components or modulators of RNase H2 substrate specificity, the assay comprising:
i) contacting the test substance with RNase H2 or a component thereof, wherein the component is RNASEH2B, RNASEH2C, RNASEH2A or any combination thereof;
ii) measuring the activity of RNase H2 or a component thereof for any time in the presence of the test substance; and iii) measuring the activity of the test substance on RNase H2 or a component thereof.

  This assay can be performed on all RNase H2 complexes, which may be the recombinant complexes described above. Alternatively, this assay can be modified and performed on components of the RNase H2 complex, which can be, for example, recombinant RNASEH2B, RNASEH2C, or RNASEH2A.

  The above-described assay may be performed by contacting a test substance with a mutant of RNase H2 or a mutant component thereof (for example, RNASEH2A, RNASEH2B, or RNASEH2C or a combination thereof). A variant of RNase H2 or a component thereof may be expressed in cell lines such as lymphoblastoid cell lines and non-human transgenic animals such as rodents.

In one embodiment, the RNase H2 complex is present intracellularly.
In one embodiment, the assay may be performed on inflammatory modulators, which may be pro-inflammatory or anti-inflammatory. In one embodiment, the assay may be performed to identify antimicrobial (eg, antiviral) agents.

  The assay may be a cell-based assay that measures endogenous RNase H2 activity. This assay can identify regulators that act indirectly on RNase H2 or pathways upstream of RNase H2. This assay is useful in measuring the effects of viral RNase H or mammalian (eg, human) RNase H2 inactivators or inhibitors, which may include such inactivators and inhibitors. This is because if the factor has specificity for only viral RNase H, it can be an effective antiviral therapy. Such an antiviral factor presumed to act on the RNase H2 complex as described herein is important in measuring the effect, particularly the range considered to be a side effect on the mammalian host in vivo.

  Step ii) of the assay can be performed, for example, by introducing a labeled oligonucleotide substrate. In one embodiment, the labeled oligonucleotide releases a fluorescent tag upon degradation with RNase H2. An oligonucleotide that has not been degraded does not fluoresce because there is a quencher molecule in the vicinity of the other base strand. Thus, fluorescence intensity is an indicator of RNase H2 activity. In this embodiment, the oligonucleotide acts as a substrate for RNase H2 or a component thereof. The oligonucleotide may be double-stranded DNA, double-stranded RNA, or a double-stranded DNA-RNA hybrid molecule.

  Step iii) can be performed by comparing to RNase H2 activity under the same conditions in the absence of the test substance.

  This assay can be performed over a period of time (ie, a kinetic assay), for example, can be performed from 10 minutes to 60 minutes. The measurement of RNase H2 activity can be carried out at an appropriate time interval (preferably at regular intervals) during the measurement, for example, at intervals of 2 to 20 minutes. In one embodiment, the assay is performed over 30 minutes and the activity measurement is performed every 5 minutes.

Furthermore, the present invention provides an assay that measures the effects of modifications (eg, amino acid mutations) on one or more components of RNase H2, the assay comprising:
i) providing RNase H2, wherein at least one component is mutated relative to the wild type;
ii) contacting said RNase H2 with its substrate;
iii) measuring the activity of the RNase H2 over an arbitrary period of time; and
iv) measuring the effect of the modified component on RNase H2 activity.

  In one embodiment, the wild type of RNASEH2B has the amino acid sequence of SEQ ID NO: 2. In one embodiment, the wild type of RNASEH2C has the amino acid sequence of SEQ ID NO: 4. In one embodiment, the wild type of RNASEH2A has the amino acid sequence of SEQ ID NO: 6.

In one embodiment, RNase H2 is recombinant.
In one embodiment, RNase H2 is expressed in a cell line such as a lymphoblastoid cell line. Alternatively, RNase H2 can be expressed in non-human transgenic animals.
In one embodiment, the RNase H2 complex is present intracellularly.

  Step ii) of the above assay can be performed, for example, by introducing a labeled oligonucleotide substrate. In one embodiment, the labeled oligonucleotide releases a fluorescent tag upon degradation with RNase H2. An oligonucleotide that has not undergone degradation does not fluoresce because there is a quencher molecule in the vicinity on the other base strand. Thus, fluorescence intensity is an indicator of RNase H2 activity. In this embodiment, the oligonucleotide acts as a substrate for RNase H2 or a component thereof. This oligonucleotide may be double-stranded DNA with embedded ribonucleotides, RNA, or a double-stranded DNA-RNA hybrid molecule.

  Step iii) can be performed by comparing to RNase H2 activity under the same conditions in the absence of the test substance.

  In one embodiment, the assay for the modified component of the RNase H2 complex is performed in the presence or absence of other components. In such embodiments, it may be beneficial to use an RNase H2 component variant that affects functionality, such as RNASEH2A (AGS4) G37S or RNASEH2B (AGS2) A177T, and the assay By doing so, recovery of RNase H2 activity can be examined. Alternatively, a decrease in RNase H2 activity can be examined by performing an assay. Alternatively, the assay can examine changes in the substrate specificity of RNase H2.

  This assay can be performed over a period of time (ie, a kinetic assay), for example, performed for 10 to 60 minutes. In that case, the measurement of RNase H2 activity can be performed at an appropriate time interval (preferably at a constant interval) during the measurement, for example, at intervals of 2 to 20 minutes. In one embodiment, the assay is performed over 30 minutes and the activity measurement is performed every 5 minutes.

  RNASEH2 knockout mice can be generated by standard techniques. After such mice were challenged with viruses according to the method performed in transgenic mice expressing IFN-α, as shown in Akwa et al., J Immunology 161: 5016-5026 (1998), Mammalian RNase H2 agonists can be used to test and optionally tested in vivo by the above-described assay to see if the effect of the virus has improved. Such knockout mice are part of the present invention.

In another aspect, the invention provides an assay for detecting a mutation in the RNASEH2B, RNASEH2C or RNASEH2A gene in a patient's genome, the assay comprising:
i) lysing white blood cells collected from the patient; and
ii) measuring RNase H2 activity of lysed white blood cells.

  The mutation to be detected is a mutation that affects RNase H2 activity that suppresses or enhances that activity relative to the normal range of the wild-type complex.

  In some cases, RNASEH2B, RNASEH2C, or RNASEH2A genes can be sequenced when a decrease or enhancement of RNase H2 activity is observed compared to the normal range.

  This assay identifies patients with reduced RNase H2 activity due to atypical or homozygous mutations in any gene of RNASEH2B, RNASEH2C, or RNASEH2A, so that AGS can be used as a diagnostic aid or for children with AGS It can be used to provide appropriate counseling for parents who are at risk of becoming a patient. This assay can also be beneficial in providing diagnostic treatment for people at high risk for autoimmune diseases, congenital viral infections and viral infections (due to reduced RNase H2 activity).

  Similarly, this assay can also identify patients with SLE and other autoimmune diseases and those at risk of having a child with SLE or other autoimmune diseases. In addition, this assay can be used to provide information on the extent of viral infections, as a diagnostic aid for viral infections, and to identify patients susceptible to microbial infections, especially viral infections. It is not limited to the disease.

  In another embodiment, this assay for measuring RNase H2 activity can be performed solely by identifying the genotype of a gene sample obtained from a patient, including fingerprinting and satellite methods. Alternatively, there is a method for specifying the base sequence of the relevant part in the genome. Obtained genetic information identifies patients with altered RNase H2 activity, diagnoses susceptibility to AGS, SLE, autoimmune diseases, and bacterial infections, or identifies genetic risks and trends It can be used as an auxiliary to

  In yet another aspect, the present invention provides a protein encoded by any one of the nucleotide sequences of SEQ ID NO: 1, 3 or 5, or a homologue thereof, a protein comprising any amino acid sequence of SEQ ID NO: 2, 4 or 6, Polyclonal or monoclonal antibodies that can specifically bind to the homologue or the recombinant RNase H2 complex described above are included. Also included are antibodies to any one variant of the RNase H2 complex component (eg, the G37S variant of RNASEH2A). A monoclonal antibody can be produced, for example, by immunizing a mouse with a recombinant RNase H2 complex having enzyme activity and then performing hybridoma fusion by a known method. Clones can be screened by ELISA and verified by immunofluorescence and Western blot assays using cells that express epitope-tagged constructs. The antibodies can be used as an aid to molecular biology techniques, for the purification of RNase H2 (eg, for diagnostic tests) or as an aid to testing the RNase H2 complex or its function.

  In one embodiment, the antibody is a therapeutic antibody and is produced in a cell line such as a hybridoma (see Kohler et al., Nature 256: 495-497, 1975 and Galfre Meth Enzymol 73: 3, 1981). Suitable therapeutic antibodies include rodent antibodies, chimeric antibodies, scFvs, humanized antibodies and human antibodies. A chimeric antibody is an antibody prepared by genetic engineering, and about one-third is composed of a non-human-derived protein and about two-thirds is composed of a human-derived protein. Humanized antibodies are genetically engineered and contain the lowest amount of non-human protein (usually 5 to 10%) in human antibodies to minimize adverse host immune responses (Riechmann et al., Nature 332: 323-327, 1988).

  Antibody fragments such as F (ab ') 2, Fab and Fv fragments also fall under "antibodies".

  The present invention also provides a diagnostic kit comprising at least one primer for RNASEH2A, RNASEH2B or RNASEH2C. Suitable primers include those shown in Table 1 and combined with appropriate auxiliary elements. Suitable auxiliary elements herein include buffers, dNTPs, enzymes (eg, TAQ polymerase), labeled compounds, and the like. The kit is used to detect genetic abnormalities in a nucleic acid sample, and the genetic abnormalities (if detected) are AGS, SLE, autoimmune diseases and bacterial infections, especially viral infections (but not A genetic tendency for susceptibility to disease).

  In one embodiment, antibodies specific for the RNase H2 complex described above can be used to identify RNase H2 containing mutant proteins.

  In another embodiment, antibodies specific for the RNase H2 complex described above can be used in therapy. For example, this antibody can be used to enhance the effects of gene therapy using viruses or to stimulate the immune response of a host.

The present invention provides for the production of a non-human transgenic animal having a genome encoding mutant RNASEH2A, RNASEH2B or RNASEH2C, wherein the sugar is:
a) introducing a recombinant gene construct comprising a polynucleotide encoding mutant RNASEH2A, RNASEH2B or RNASEH2C into a non-human zygote or non-human embryonic stem cell;
b) creating a non-human transgenic animal from said zygote or embryonic stem cell;
c) producing a non-human transgenic animal having a genome encoding a mutant RNASEH2A, RHASEH2B or RNASEH2C.

  The polynucleotide sequence encoding the mutant RNASEH2A, RHASEH2B or RNASEH2C is preferably linked to a promoter so that the protein is expressed in the transgenic animal.

  A non-human transgenic animal having a genome encoding mutant RNASEH2A, RHASEH2B or RNASEH2C is a further aspect of the present invention.

  The non-human transgenic animal may be any suitable animal, and suitable examples include mice, rats, primates, hamsters, rabbits, dogs, cats, fish, cows, pigs and sheep. However, this list is not exhaustive and other animals can be used.

  In one embodiment, the non-human transgenic animal is a rodent. Suitable examples include rats and mice.

  Embryonic stem cells used in known techniques that may be used in the methods of the invention include embryonic stem cells obtained from C57BL / 6, CBA /, BALB / c, DBA / 2 and SV129 mouse lineages However, the present invention is not limited to this. Preferably, embryonic stem cells derived from C57BL / 6 mice are used (Seong, E et al., Trends Genet. 20, 59-62, 2004; Wolfer et al., Trends Neurosci. 25: 336-340, 2002).

  The transgene (including the coding gene sequence of RNASEH2A, RNASEH2B or RNASEH2C) can be introduced into the germline of an animal by a variety of methods. For example, the transgene can be directly injected into the male pronucleus of a fertilized egg (see, eg, Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor Press (1994)). As a result, different numbers of copies of the transgene are introduced randomly at one locus, usually head-to-tail (see, for example, Costantini and Lacy, Nature, 294, 92, 1981). Injected eggs are transplanted to pseudopregnancy-induced recipient females. Some offspring so born may have integrated one or more copies of the transgene into the genome, usually one site. These founder animals are crossed to create a transgenic animal line and backcrossed to create a system with a genetic background to select. One skilled in the art will appreciate the benefits of introducing a transgene into both chromosomes. Alternatively, the transgene can be introduced into the animal by gene targeting, such as homologous recombination in embryonic stem (ES) cells. A known method suitable for introducing a transgene is blastocyst transfer. In this method, a targeting construct containing the transgene is prepared by methods known in the art.

  The targeting construct containing the transgene can be introduced into an appropriate host cell by a known method. Transgenic embryo production and screening thereof can be performed by known techniques described in, for example, Joyner ed., Gene Targeting, A Practical Approach, Oxford University Press, 1993. Screening of DNA of transgenic animals or embryos can be performed by Southern blotting or RCR.

  The non-human transgenic animal may be a healthy animal or may show signs of a disease or disorder due to a mutation introduced by the transgene. Such transgenic animals are suitable for pharmacological research on drugs, and can be used as disease models for AGS, SLE, autoimmune diseases and viral infections, for example. Such models are more susceptible to AGS, SLE, autoimmune diseases and viral infections.

  The invention is further illustrated by the following non-limiting examples and figures.

Example 1: Patients and subjects All patients in this study met the diagnostic criteria for AGS, had neurological features indicative of early brain damage, and no evidence of infection of general infections before childbirth Intracranial calcification was seen in a typical area with cerebrospinal fluid (CSF) lymphocytosis (> 5 cells / mm ³ ) or IFN-α> 2 IU / mL in CSF. With consent, blood samples were taken from sick children, their parents and unaffected siblings, and genomic DNA was extracted from peripheral blood leukocytes by standard methods. This study was conducted with the approval of the Leeds Health Authority / United Teaching Hospitals NHS Trust Research Ethics Committee and Scottish Multicentre Research Ethics Committee (04: MRE00 / 19).

Genotyping and linkage analysis Genome-wide scans with SNP arrays were performed using the Affymetrix Human Mapping 10K Xba142 2.0 GeneChipsR from MRC Geneservice (Cambridge, UK). Established microsatellite markers selected from Marshfield's genetic map (http://research.marshfieldclinic.org/genetics) according to previously reported methods (Jackson et al., Am J Hum Genet 63: 541-546 (1998)) And high density genotyping of the AGS2 and AGS3 loci using a new microsatellite identified by the human genome browser sequence (May 2004, http://genome.ucsc.edu/). Linkage analysis was performed using GENUNTER (2.0β) (Kruglyak et al., Am J Hum Genet 56: 1347-1363 (1996)), autosomal recessive inheritance model, disease allele frequency 1 to 100, penetrance Was carried out on the assumption that the allele frequencies of the markers were equal.

Bioinformatics database search uses PSI-BLAST (http://www.ncbi.nlm.nih.gov/blast/, Altschul et al. Nucleic Acids Res. 25: 3389-3402 (1997)). The inclusion threshold was 2 × 10 ⁻³ , but this database is a non-redundant protein sequence database.

  Protein structural analysis is performed using the default setting by the competitive modeling method using SWISSMODEL (Schwede et al. Nucleic Acids Res 31: 3381-3385 (2003)), and WHAT-CHECK (Hooft et al. Nature 381: 272 (1996)), the accuracy of the predicted structure of the RNASEH2A protein (NP-006388) was confirmed. As the predictive template, four archaea RNase H2 homologous proteins 1uaxA, 1io2A, 1x1pA and 1keB with known structures, which are the best available, were used. The active site and predicted substrate binding site (Chapados et al., J Mol Biol 307: 541-556 (2001)) is VMD (V1.8.3) (Humphrey et al., J Mol Graph 14: 33- 38, 27-28 (1 996)).

Detection of mutation sites Primers were designed to amplify the coding exons of RNASEH2B, RNASEH2C and RNASEH2A (primer sequences are shown in Table 1). The sequence of the purified PCR amplification product was determined by electrophoresis using a dye terminator (Applied Biosystems) and an ABI 3700 capillary sequencer (Applied Biosystems) or Megabace 500 capillary sequencer (Amersham Pharmacia). Mutation analysis was performed using Mutation Surveyor (Softgenetics).

Vector production The Gateway vector system (Invitrogen) was used for the production of mammalian expression vectors. The coding regions of RNASEH2B, RNASEH2C and RNASEH2A were amplified from the plasmid clones (CSODF031YM15, CR6022872 (Invitrogen) and clone IRAup969G0361D, BC011748 (RZPD), respectively) and introduced into pDONR221 ?. For AYP1, a commercially available pENTRY vector (clone IOH27907, Human Ultimate? Full ORF Gateway Shuttle Clone, NM_032193, Invitrogen) was used. pcDNA3.1mychis-Dest and pCGT-Dest used the Gateway Vector Conversion System to transfer the Gateway reading frame cassette from pcDNA3.1mychis (Invitrogen) and pCGT (Van Aelst et al., 3786). ) And inserted into the multiple cloning site. Site-directed mutagenesis was performed using the RNASEH2A pENTRY clone and using the Stratagene Quikchange kit according to the manufacturer's instructions.

Immunoprecipitation and Western blotting For HEK293T cells, Lipofectamine (Invitrogen) was used according to the manufacturer's instructions and 1 μg of each construct was transiently co-introduced. 24 hours later 4 with 50 mM Tris (pH 7.8), 280 mM NaCl, 0.5% NP40, 0.2 mM EDTA, 0.2 mM EGTA, 10% glycerol, 0.1 mM sodium orthovanadate, 1 μM DTT and 1 μM PMSF Cells were lysed for 10 minutes at ° C. Lysed cells were diluted 1: 1 with 20 mM Hepes (pH 7.9), 10 mM KCI, 1 mM EGTA, 10% glycerol and 0.1 mM sodium orthovanadate and centrifuged after 10 minutes (15800 g, 10 minutes, 4 ° C.) and the extract was collected. 1 μg mouse anti-myc antibody (clone 9B11, Cell Signaling) or 1 μg mouse IgG (Santa) using Protein A / G PLUS agarose (Santa Cruz) according to the manufacturer's instructions for 500 μg of cell lysate containing protein Immunoprecipitation was performed by Cruz). For Western blot, mouse anti-myc monoclonal antibody was used at a concentration of 1/1000 and mouse anti-T7 monoclonal antibody (Novagen) at a concentration of 1/5000.

RNase H assay Annealing was performed by denaturing 10 μM oligonucleotide (Eurogentec) in 60 mM KCI, 50 mM TrisHCl pH 8 at 95 ° C. for 5 minutes and then gradually returning to room temperature. The RNase H activity fluorescence assay was performed by shaking 100 μL containing 60 mM KCI, 50 mM TrisHCl pH 8, 10 mM MgCl ₂ and 0.25 μM oligonucleotide duplex in a 96-well flat-bottom plate on an orbital shaker at 37 ° C. for 3 hours at 60 rpm. I went there. Each reaction uses 1/10 the amount of immunoprecipitate and 2.5 units of E. coli as a positive control. E. coli RNase H (Invitrogen) was used. Fluorescence was measured for 100 msec using a Victor ² 1420 multilabel counter (Perkin Elmer) with an excitation filter of 480 nm and a transmission filter of 535 nm.

Results AGS2 locus refinement and identification of RNASEH2B (FLJ11712) High-density genotyping of microsatellite markers was performed on 10 families to purify the AGS2 locus. A small region was found in two non-cognate families (F8 and F10, FIG. 7) showing overlapping of homozygous markers. Such regions are thought to represent homozygous chromosomal regions (Broman et al., Am J Hum Genet 65: 1493-1550 (1999) and Gibson et al., Hum Mol Genet 15: 789-795 (2006)), in the case of rare recessive genetic diseases, it is very likely to contain disease genes (Lander et al., Science 265: 2049-2054 (1987)). By using this overlapping region of homozygous markers, the AGS2 essential region could be purified to a 571 kbp region between the gene markers AC13778890TG19 and D13S788 (FIG. 2) on chromosome 13q14.3. Sequences were identified for coding exons of all four annotated genes in the essential region.

RNASEH2B (FLJ11712) is an AGS2 gene Although a missense mutation of FLJ11712 was found in 7 of the families screened, no pathogenic mutation was found in DLEU7, GUCY1B2, or FLJ30707. When mutation screening of FLJ11712 was performed in a larger cohort, a total of 18 families showing mutations in this gene were found (Table 2, FIG. 2). Mutations were mostly missense and two were frequently seen in different ethnic groups (A177T, V185G). All missense mutations are non-conservative substitutions of a mammalian conserved residue (FIG. 8), with the exception of a single conservative residue mutation (Y219H) that goes back to Dictostelium. Saved. Nonsense mutations were found in two families, the exon 2 stop codon (F17) and the intron 6 splice donor mutation (F15). In both cases, the person with the disease was a synthetic heterozygote and the second mutation was a missense mutation. From the above, according to the observed mutation spectrum, it is considered that the influence of the mutation is not a complete loss of the FLJ11712 protein but a hypomorphic type. In all families, the mutation was associated with disease and all parents were heterozygous for this mutation. For each mutation, at least 160 control alleles were genotyped. Only one of the most common mutations, A177T, was found to be heterozygous in the 241 samples tested.

RNASEH2B (FLJ11712) is an orthologue of yeast Rnh2Bp FLJ11712 encodes a protein of 308 amino acids, but this protein has so far been unclear in function. The semi-quantitative RT-PCR method has detected that FLJ11712 is detected in a wide range of human tissues and expressed ubiquitously (data not shown).

When database search using PSI-BLAST (Altschuhl et al., Nucleic Acids Res 25: 3389-3402 (1997)) was repeated four times, human FLJ11712 and Saccharomyces cerevisiae protein Rnh2Bp (Rnh202) were used. There was considerable similarity (E = 4 × 10 ⁻⁵ ) (FIG. 8). Rnh2Bp is an essential component of the yeast RNase H2 enzyme complex (Jeong et al., Nucleic Acids Res 32: 407-414 (2004)). This finding suggests that FLJ11712 is functioning in a similar complex in mammals, suggesting that in the case of AGS, another RNase H component may also be mutated.

RNASEH2C (AYP1) is an AGS3 gene SNP array genome scan was performed on 6 non-cognate families consisting of 5 Pakistani families (F30-34) and 1 Bangladesh family (F35). This scan and subsequent microsatellite genotyping (FIG. 9) identified a new locus AGS3 between markers D11S4205 and D11S987 on chromosome 11q13.2, which has a maximum multipoint LOD value of 66.8 cM and 4 .54 (Marshfield gene map). Furthermore, genotyping in a 5-Pakistani family suggested the presence of an ancestor haplotype (genotype shown in bold, FIG. 9), but this and the small homozygous region in the F30 family also allowed the AGS3 essential region to be identified as D11S4205. It was possible to purify into a 4.9 cm section between D11S987.

  Of note, AYP1, which encodes a protein that is biochemically co-purified with human RNase H2A (Frank et al., Proc Natl Acad Sci USA 95: 12872-12877 (1988)), is within this essential region. (FIG. 3). Therefore, the sequence of AYP1 was determined and non-conservative missense mutations in these 6 families were identified (see Table 3 and FIG. 9). A homozygous mutation at codon 69 (R69W) was found in all patients of a 5-Pakistani family sharing a gene believed to be an ancestral haplotype. Another homozygous mutation, K1431, was found in the Bangladesh family. This mutation was associated with disease within the family, and none of the mutations were found in at least 172 Asian control alleles. RT-PCR expression profiling of AYP1 showed a broad expression pattern similar to FLJ11712.

  At the same time, the inventors have identified S. aureus in which AYP1 is the second subunit of yeast RNase H2. cerevisiae Rnh2Cp (Jeong et al., Nucleic Acids Res 32: 4407-414 (2004)). Human AYP1 and S. cerevisiae were obtained by four search iterations of database search with the Kluyveromyces walti open reading frame. cerevisiae Rnh2Cp was identified (FIG. 8).

RNASEH2A is an AGS4 gene RNASEH2A is a 7 kbp exon 8 gene on chromosome 19p13.13, which encodes a protein consisting of 299 amino acids. RNASE2A did not colocalize with any AGS locus from which the genetic map was generated. However, review of SNP array genome scan data reveals homozygosity for the RNASE2A locus of a non-cognate AGS family (Sanchis et al., J Pediatr 146: 701-705, 2005) with a previously reported Spanish Caucasian ancestry A small area was found (Figure 4a). Examining the sequence for two diseased children in this family c. A homozygous single base conversion of 109G → A was observed, resulting in a non-conservative missense mutation of G37S (FIG. 4b). This amino acid is conserved in a wide range from human to archaea (Fig. 4c), located at the bent portion of the end of the first β-sheet at the bottom of the substrate-binding groove and in the vicinity of the RNase H active site. (Fig. 4d) (Chapados et al., J Mol Biol 307: 541-546, 2001). This mutation was not detected in the 178 Caucasian control allele, and the parents were heterozygous for this mutation.

RNASEH2B (FLJ11712), RNASEH2C (AYP1) and RNASEH2A form a complex with RNase H2 activity in vitro. In view of sequence homology with the cerevisiae Rnh2Ap-Rnh2Bp-Rnh2Cp complex, RNASEH2B, RNASEH2C and RNASEH2A proteins are thought to form protein complexes with RNase H2 activity (FIG. 5a). In order to verify this, three genes were cloned using the Gateway system, introduced into mammalian expression vectors (pCGT-Dest (T7) and pCDNA3.1mychis-Dest) with epitope tags, and these three types of constructs were introduced. Was transiently co-introduced into HEK293 cells. Immunoprecipitation of C-terminally tagged FLJ11712-myc with anti-myc antibody revealed N-terminal T7-tagged AYP1 and T7-tagged RNASE2A pulldown (FIG. 5b), and these three subunits interacted in vitro. It was confirmed to have.

  The enzyme activity of this complex was examined by applying a previously reported fluorescence measurement method (Parniak et al., Anal Biochem 33-39, 2003). The fluorescently labeled oligonucleotide was DABCYL labeled and annealed with a complementary DNA oligonucleotide that quenches fluorescence (FIG. 5c). When the enzymatic cleavage of the oligonucleotide bound to the fluorescent dye occurs, the fluorescent dye is released from the adjacent quenching molecule and a fluorescent signal is generated (FIG. 5c).

  The inventors found that "oligo C", an RNA: DNA duplex, was effectively cleaved by the immunoprecipitation complex, and it was confirmed that this complex exhibits RNase H activity. (FIG. 5d). Furthermore, this immunoprecipitated complex recognizes a single ribonucleotide embedded in the DNA-DNA duplex and is a “type 1” RNase H E. coli. In contrast to E. coli RNase H, it effectively cleaves “oligo B”, indicating that it has the required “type 2” RNase H activity. As expected, “Oligo A” did not undergo cleavage because it was synthesized using a nuclease resistant 2-O ′ methyl RNA structure.

Mutation of RNase H2 complex reduces enzyme activity Selected pathogenic mutations (FLJ11712, A177T, T1631; AYP1, R69W, K143I; RNASEH2A G37S) were introduced using site-specific mutagenesis of each gene and HEK293 Transiently introduced into cells and expressed. Immunoprecipitation was performed to examine the effect on the stability of the complex and an enzyme activity assay was performed (FIGS. 6a and 6b). These experiments showed that the RNASEH2A G37S mutation did not affect the stability of the complex, but the enzyme activity was significantly reduced as expected for the mutation at the catalytic site (FIG. 6b). . In the case of the AYP1 mutation, the amount of AYP subunit that forms a complex with other subunits and causes pull-down decreased. This decrease was associated with a decrease in enzyme activity. This indicates that the decrease in enzyme activity is the result of inhibition of complex formation by mutation.

Example 2: RNase H2 activity assay of poly (I: C) treated HCT116 cells According to the inventors' observations, innate immunity was inappropriately activated in AGS, indicating that RNase H2 is It is shown to be involved in the innate immune response. In order to perform this verification, poly (I: C), which is a synthetic analog of dsRNA, was treated, but this synthetic analog is associated with viral infection, and dsRNA-induced protein kinase (PKR) or toll-like receptor 3 (toll) It is known to stimulate innate immune responses by inducing cytokine production via like receptor 3). RPMI1640 medium containing 10% FCS and 1% penicillin / streptomycin over time with 25 μg / mL poly (I: C) (Invivogen, USA) against semi-confluent HCT116 colon cancer cells The process was performed. A cell lysate containing protein was prepared from the cells, and an RNase H2 fluorescence assay was performed.

  The results are shown in FIG. The enzyme activity doubled after 2 hours, suggesting that RNase H2 is located downstream of this pathway and may be an effector of the innate immune response.

Example 3: Polyclonal antibody Polyclonal antibodies were generated by expressing RNase H2 immunogen (immunogen) as a bacterially expressed protein ("Antibodies: A Laboratory Manual" edited by Harlow and Lane, Cold Spring Harbor Laboratory Press (1988) (Issued December 1) ISBN 978-0879693145). This expressed protein was purified by GST tag attached to the B subunit. The GST tag was then cleaved with PreScission protease (Amersham) and the resulting protein was injected into a sheep (Eurogentec). Freund's adjuvant was used at the time of injection. For the first injection, complete adjuvant was used followed by boosting with incomplete adjuvant. The resulting sheep serum was passed through a column bound with an antigen complex and purified using immunoaffinity. This antigen complex was the same as that used as the immunogen. Western blots (see FIG. 11) show that the antibody can detect overexpressed proper molecular weight subunits. That is, the antibody was shown to have specificity for the A, B and C subunits of RNase H2. Another band (*) may indicate one of the endogenous proteins. The band obtained by Western blotting was identified by reprobing with an anti-tag antibody.

The immunogenic sequences used are as follows:
For anti-RNASEH2B (including linker residues left from protease cleavage)
LEVLFQGPLGSPEFPSTSLYKKAGSTMAAGVDCGDGVGARQHVFLVSE YLKDASKKMKNGLMFVKLVNPCSGEGAIYLFNMCLQQLFEVKVFKEKHH SWFINQSVQSGGLLHFATPVDPLFLLLHYLIKADKEGKFQPLDQVVVDNV FPNCILLLKLPGLEKLLHHVTEEKGNPEIDNKKYYKYSKEKTLKWLEKKVN QNAALKTNNVNVSSRVQSTAFFSGDQASTDKEEDYIRYAHGLISDYIPK ELSDDLSKYLKPEPSASLPNPPSKKIKLSDEPVEAKEDYTKFNTKDLKTE KKNSKMTAAQKALAKVDKSGMKSIDTFFGVKNKKKIGKV (SEQ ID NO: 52).

For anti-RNASEH2C
MESGDEAAIERHRVHLRSATLRDAVPATLHLLPCEVAVDGPAPVGRFFT PAIRQGPEGLEVSFRGRCLRGEEVAVPPGLVGWMVTEEKKVSMGKPD PLRDSGTDDQEEEPLERDFDRFIGATANFSRFTLWGLETIPGPDAKVRG ALTWPSLAAAIHAQVPED (sequence number 53).

For anti-RNASEH2A
MDLSELERDNTGRCRLSSPVPAVCRKEPCVLGVDEAGRGPVLGPMVYA ICYCPLPRLADLEALKVADSKTLLESERERLFAKMEDTDFVGWALDVLSP NLISTSMLGRVKYNLNSLSHDTATGLIQYALDQGVNVTQVFVDTVGMPE TYQARLQQSFPGI EVNKAKADALYPVVSAASICAKVARDQAVKKWQFV EKLQDLDTDYGSGYPNDPKTKAWLKEHVEPVFGFPQFVRFSWRTAQTIL EKEAEDVIWEDSASENQEGLRKITSYFLNEGSQARPRSSHRYFLERGLE
SATSL (SEQ ID NO: 54).

Example 4: Lymphoblastoid cell line (LCL)
Lymphoblast-like cell line (LCL) was created as a cell line in which primary B cells of AGS patients were infected with EBV and continuously proliferated. Suitable methods for making LCL are known and described, for example, in Penno et al., Methods in Cell Science 15 (1): 43-47, 1993. LCL was transformed to show representative mutations found in patient subunits.

The following LCL was made.

  Cellular enzyme activity can be assayed by the previously described RNase H activity fluorometry. In addition to the endpoint assay (Figure 13), kinetic assays can also be measured at regular time intervals (eg every 5 minutes) with a Perkin Elmer Victor Ill type instrument with a built-in temperature controller (set at 37 ° C) . An example of assay for examining the activity of the RNase H2 mutant lymphoblastoid cell line is shown in FIG.

Example 5: Recombinant RNase H2
A recombinant RNase H2 protein complex having enzymatic activity was synthesized and purified using a polycistronic bacterial expression construct containing 3 genes (see FIG. 15a). The activity of the mutant and wild type complexes can be measured by enzyme assay, as shown in FIG. 15b.

Example 6: RNase H2 suppresses viral replication
RNA-DNA intermediates are essential for viral replication. Therefore, upregulation of endogenous RNase H2 activity can be expected to be beneficial as a host reaction to suppress viral replication. The inventors have obtained evidence to support this idea, and enzyme activity increases within hours after treatment of HCT116 cells with dsRNA (poly: C), a potent activator of innate immune signals (see FIG. 10). ).

Claims (61)

A recombinant of RNase H2,
i) a protein encoded by a base sequence consisting of one of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or a homologue thereof;
ii) A recombinant of RNase H2, characterized by comprising a protein consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 2 or SEQ ID NO: 6, or a homologue thereof. The RNase H2 recombinant according to claim 1, comprising a protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 or a homologue thereof. The RNase H2 recombinant according to claim 1, comprising a protein encoded by the nucleotide sequence shown in SEQ ID NO: 3 or a homologue thereof. The RNase H2 recombinant according to claim 1, comprising a protein encoded by the nucleotide sequence shown in SEQ ID NO: 5 or a homologue thereof. The RNase H2 recombinant according to claim 1, comprising a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a homologue thereof. The RNase H2 recombinant according to claim 1, comprising a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or a homologue thereof. The RNase H2 recombinant according to claim 1, comprising a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or a homologue thereof. A recombinant of RNase H2,
i) RNASEH2B encoded by the nucleotide sequence of SEQ ID NO: 1 or a homolog thereof or RNASEH2B having the amino acid sequence of SEQ ID NO: 2 or a homolog thereof,
ii) RNASEH2C encoded by the nucleotide sequence of SEQ ID NO: 3 or a homolog thereof or RNASEH2C having the amino acid sequence of SEQ ID NO: 4 or a homolog thereof; and iii) RNASEH2A encoded by the nucleotide sequence of SEQ ID NO: 5 or a homolog thereof An RNase H2 recombinant comprising at least one RNASEH2A having 6 amino acid sequences or a homologue thereof. 9. The RNase H2 recombinant of claim 8, comprising at least two of i), ii) and iii). 9. The RNase H2 recombinant of claim 8, comprising each component of i), ii) and iii). A recombinant polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3, or 5 or a homologue thereof. A protein encoded by the polynucleotide of claim 11. A protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6 or a homologue thereof. A polynucleotide encoding the protein of claim 13. A vector comprising the polynucleotide of claim 11 or 14. A host cell into which the vector of claim 15 has been introduced. A lymphoblastoid cell line (LCL) expressing a variant of RNase H2. 18. The cell line of claim 17, wherein the RNase H2 has an A177T mutation of RNase H2B. 18. The cell line of claim 17, wherein the RNase H2 has a G37S mutation of RNase H2A. The host cell of claim 16 which is an ES cell line. An antibody specific for the protein of any of claims 12 and 13. The antibody of claim 21, which specifically binds to a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or a protein encoded by the polynucleotide of SEQ ID NO: 1. The antibody of claim 21, which specifically binds to a protein comprising the amino acid sequence shown in SEQ ID NO: 4 or a protein encoded by the polynucleotide of SEQ ID NO: 3. The antibody of claim 21, which specifically binds to a protein comprising the amino acid sequence shown in SEQ ID NO: 6 or a protein encoded by the polynucleotide of SEQ ID NO: 5. The antibody according to any one of claims 21 to 24, which is a polyclonal antibody. The antibody according to any one of claims 21 to 24, which is a monoclonal antibody. 27. The antibody according to any one of claims 21 to 26, which is a humanized antibody. 28. The antibody according to any one of claims 21 to 27 for use in therapy. An assay for identifying activators or inactivators of RNase H2 or its components or modulators of RNase H2 substrate specificity comprising:
i) contacting the test substance with the RNase H2 recombinant or a component thereof, wherein the component is RNASEH2B, RNASEH2C, RNASEH2A, or any combination thereof;
ii) measuring the activity of RNase H2 or a component thereof at any time in the presence of a test substance; and
iii) an assay comprising measuring the activity of the test substance against RNase H2 or a component thereof. 30. The assay of claim 29, wherein step ii) is performed for a predetermined period, and RNase H2 activity is measured periodically during that period. 31. The assay according to any one of claims 29 and 30, wherein the recombinant RNase H2 complex is present in step i). 32. The assay of claim 31, wherein said RNase H2 complex is present in a cell. 31. The assay according to any one of claims 29 and 30, wherein one of RNASEH2A, RNASEH2B and RNASEH2C is present in step i). 34. The assay according to any one of claims 29 to 33, wherein the activity of RNase H2 or a component thereof is measured in step ii) by introducing a labeled oligonucleotide. An assay for identifying activators or inactivators of RNase H2 or its components or modulators of RNase H2 substrate specificity comprising:
i) contacting the test substance with mutant RNase H2 or a mutant component thereof, wherein the component is RNASEH2A, RNASEH2B, RNASEH2C or any combination thereof;
ii) measuring the activity of the RNase H2 or a component thereof at any time in the presence of the test substance; and
iii) an assay comprising measuring the activity of the test substance against RNase H2 or a component thereof. 36. The assay of claim 35, wherein step iii) is performed for a period of time and RNase H2 activity is measured periodically during that period. 37. The assay according to any one of claims 35 and 36, wherein said RNase H2 is recombinant. 37. The assay according to any one of claims 35 and 36, wherein said RNase H2 is expressed from a lymphoblastoid cell line. 37. The assay according to any one of claims 35 and 36, wherein said RNase H2 is expressed by a non-human transgenic animal. 37. The assay according to any one of claims 35 and 36, wherein the RNase H2 complex is present intracellularly. An assay for measuring the effect of modification on one or more components of RNase H2, comprising:
i) providing RNase H2 wherein at least one component is mutated relative to the wild type;
ii) contacting said RNase H2 with a substrate for it,
iii) measuring the activity of the RNase H2 over an arbitrary period of time; and
iv) An assay comprising measuring the effect of a modified component on RNase H2 activity. 42. The assay of claim 41, wherein step iii) is performed for a period of time, and RNase H2 activity is measured periodically during that period. 43. The assay according to any one of claims 41 and 42, wherein said RNase H2 is expressed from a lymphoblastoid cell line. 43. The assay according to any one of claims 41 and 42, wherein said RNase H2 is expressed by a non-human transgenic animal. 43. The assay according to any one of claims 41 and 42, wherein said RNase H2 complex is present in a cell. An assay for detecting a mutation in the RNASEH2B, RNASEH2C or RNASEH2A gene in a patient's genome, comprising:
i) lysing leukocytes collected from a patient;
ii) an assay comprising measuring RNase H2 activity of lysed white blood cells. A method for assisting diagnosis of susceptibility to or susceptibility to SLE, AGS, autoimmune disease, bacterial infection, and genotyping a patient's RNASEH2A, RNASEH2B or RNASEH2C gene, A method characterized by comparing with. 48. The method of claim 47, wherein the bacterial infection is a viral infection. A method for producing a non-human transgenic animal having a genome encoding a mutant RNASEH2A, RNASEH2B or RNASEH2C comprising:
a) introducing a recombinant gene construct comprising a polynucleotide sequence encoding mutant RNASEH2A, RNASEH2B or RNASEH2C into a non-human zygote or non-human embryonic stem cell;
b) producing a non-human transgenic animal from said zygote or embryonic stem cell; and
c) A method for producing a transgenic animal, comprising the step of producing a non-human transgenic animal having a genome encoding a mutant RNASEH2A, RHASEH2B or RNASEH2C. 50. The method of claim 49, wherein said transgenic animal is a rodent. 51. The method of claim 50, wherein the transgenic animal is a mouse or a rat. A non-human transgenic animal having a mutated gene of RNASEH2A, RHASEH2B or RNASEH2C. 53. The transgenic animal of claim 52, having cells expressing RNASEH2A, RHASEH2B, or RNASEH2C. 54. The transgenic animal according to any one of claims 52 and 53, which is a rodent. 55. The transgenic animal of claim 54 which is a mouse or a rat. 54. Use of the transgenic animal according to any one of claims 52 and 53 as a model of AGS, SLE, autoimmune disease or bacterial infection. A diagnostic kit for diagnosing whether a patient has an AGS, SLE, prevalence of autoimmune disease or bacterial infection or a tendency or risk thereof, in combination with an appropriate adjuvant, RNASEH2A, RNASEH2B or A diagnostic kit comprising a primer for RNASEH2C. 58. The diagnostic kit of claim 57, wherein the bacterial infection is a viral infection. An assay for identifying a factor having proinflammatory activity, comprising:
i) contacting the test substance with the RNase H2 recombinant or a component thereof, wherein the component is RNASEH2B, RNASEH2C, RNASEH2A or any combination thereof; and
ii) an assay comprising measuring the activity of RNase H2 or a component thereof at any time in the presence of a test substance. 61. The assay of claim 60, wherein the factor is an anti-inflammatory factor. 61. The assay of claim 60, wherein the factor is a pro-inflammatory factor.