KR101372632B1 - Method for analysis of xpg endonuclease activity - Google Patents

Abstract

The present invention relates to a method for quantitatively analyzing the activity of XPG endonucleases. According to the present invention, XPG endonuclease activity can be analyzed simply and inexpensively without the need for overexpression or purification of the recombinant protein.

Description

JPH endonuclease activity assay method METHOD FOR ANALYSIS OF XPG ENDONUCLEASE ACTIVITY

The present invention relates to a method for analyzing the activity of XPG endonucleases.

Nucleotide excision repair (NER) pathways are thought to be the major DNA repair systems for removing bulky chemical adducts and UV photolysis products. In humans, deficiency of the major enzyme of the NER pathway, XPG (xeroderma pigmentosum (XP) of the complementation group G), leads to the development of cancer-prone syndromes and high skin cancers due to acute UV sensitivity. . This shows that XPG proteins play an important role in DNA repair and thus in maintaining genetic stability.

XPG proteins are structure specific endonucleases that cleave substrates of defined polarity during NER (nucleotide excision repair). Existing in vitro studies of the catalytic activity of purified human XPG have shown that XPG-compatible substrates can contain artificial DNA structures (bubble, splayed arms, stem-loops, Flap substrates) (A. O'Donovan, AA Davies, JG Moggs, SC West, and RD Wood, Nature 371 (1994) 432-435; E. Evans, J. Fellow, A.). Coffer, and RD Wood, EMBO. 16 (1997) 625-638). XPG protein has a molecular weight of 135 kDa and belongs to the structure specific Fen1 (Flap endonuclease 1) group. Within the N-terminal and internal regions, XPG shares similar sequences with other nuclease groups, including bacteriophage T4 RNase H. At the active site of RNase H, which can chelate two enzyme magnesium (Mg) ions, there is a conserved acidic residue. These same acidic residues in XPG may be involved in hydrolysis and thus may be involved in XPG-DNA binding.

During NER, a series of biochemical processes mediate recognition of damaged bases, cleavage of ˜26-30 nucleotides, removal of damaged paths, filling gaps and ligation. In the cleavage step, the dual incision of the opposite pole near the junction of the unpaired double stranded DNA is catalyzed by two different endonucleases. In humans, XPG protein and ERCC1-XPF complex mediate 3'- and 5'- cleavage, respectively, in double cleavage. Both endonucleases show structural specificity for the model DNA substrate.

The 3'- and 5'-ends of the damaged DNA strand are double cleaved by the XPG protein and the ERCC1-XPF complex, respectively. On the other hand, when the conventional 5'-end oligo labeling is applied, the 5'-end transfer activity of the ERCC1-XPF complex hides the 3'-cutting activity of XPG. Therefore, as another approach to solve this problem, we can consider the 3'-terminal oligo labeling method using intracellular protein extracts. This method eliminates the need for overexpression and purification of the target XPG as a prerequisite for XPG analysis.

The inventors continued to study the invention to develop a simple and accurate XPG endonuclease activity assay method applicable to both cells and tissues.

[Prior Art Literature]

[Non-Patent Document]

A. O'Donovan, A.A. Davies, J.G. Moggs, S.C. West, and R.D. Wood, Nature 371 (1994a) 432-435

E. Evans, J. Fellow, A. Coffer, and R.D. Wood, EMBO. 16 (1997) 625-638)

Y. Habraken et al. (A. Constantinou, D. Gunz, E. Evans, P. Lalle, PA Bates, RD Wood, and SG Glarkson, J. Biol. Chem. 274 (1999) 5637-5648

It is an object of the present invention to provide a new method for analyzing the endonuclease activity of XPG.

Another object of the present invention is to provide a kit for analyzing endonuclease activity of XPG.

According to the present invention,

Preparing a biological extract sample containing xeroderma pigmentosum (XP) of the complementation group G;

Preparing a DNA bubble substrate;

Attaching a detectable label to the 3'-end of the DNA bubble substrate to form a 3'-end labeled DNA bubble substrate; And

Mixing the biological extract sample with the 3'-terminal labeled DNA bubble substrate;

Provided is an XPG endonuclease activity analysis method comprising the step.

The biological extract sample may be a cell nuclear extract, a cell total protein extract or a tissue total protein extract.

The DNA bubble substrate may be complementary to oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2.

The detectable label may be a radioisotope, fluorescent material, luminescent material or precursor of a luminescent material, specifically ³² P, ³⁵ S, ¹³¹ I, ¹²³ I, ¹²⁵ I, ³ H, carboxyfluorescein (FAM) , Tetramethyldamine (TAMRA), Cy3, Cy5, IRDye series, fluorescein (fluorescein), fluorescein isocyanate (FITC), rhodamine, Texas red, Alexa series, Dioxygeninine (DIG) and biotin (biotin) may be selected from, but is not necessarily limited thereto.

The method may further include identifying that the DNA bubble is cleaved after mixing the biological extract sample with the 3'-terminal labeled DNA bubble substrate.

The size of the cleaved DNA bubble may be 30 nucleotides or less.

Mixing of the biological sample and the DNA bubble substrate can be performed in the presence of a reaction buffer.

The reaction buffer may have a pH of 6.0 to 8.5.

The reaction buffer is MgCl ₂ Or MnCl ₂ .

The amount of MgCl ₂ or MnCl ₂ added may be 2-10 mM.

In another aspect, the present invention provides a kit for analyzing XPG endonuclease activity comprising the above-mentioned biological extract and a DNA bubble substrate.

The present invention analyzes the activity of the endonuclease of XPG directly from biological extracts containing XPG rather than purified XPG protein, thus preparing recombinant proteins for overexpression of XPG protein, isolation of XPG protein, purification process, and the like. The analysis is simple without the need for additional steps. In addition, there is little possibility of protein denaturation by the separation and purification steps of the protein in the analysis process, the XPG endonuclease activity can be analyzed close to the actual biological activity.

In addition, the activity of XPG endonucleases can be efficiently evaluated by using a nucleotide bubble substrate, ie, a DNA bubble substrate, suitable as a substrate of XPG protein. In particular, the XPG endonuine is used by using a bubble substrate with a detectable label at the 3′-end to exclude the effect of the 5′-terminal cleavage activity of the ERCC1-XPF complex, another endonuclease involved in the double cleavage of NER. The 3 'terminal cleavage activity of the clease can be accurately assessed.

The biological extract sample may be a cell nuclear protein extract, a cell total protein extract or a tissue total protein extract. These protein extracts contain XPG. When the biological extract and the DNA bubble substrate are mixed, the endonuclease XPG is in contact with the DNA bubble substrate to exhibit cleavage activity.

That is, as shown in FIG. 1A, the XPG cuts the 3 'end of the bubble structure and the ERCC1-XPF complex (denoted XPF) cuts the 5' end of the bubble structure around the bubble structure of the bubble substrate. Usually, XPG and ERCC1-XPF complexes are known to cut 3 to 5 bases in the 3 'and 5' directions, respectively, around the C repeat sequence. Therefore, in FIG. 1A, when the 5'-end labeled bubble substrate is used, not only XPG and ERCC1-XPF complexes cleave simultaneously but also XPG does not have cleavage activity and only ERCC1-XPF complex shows cleavage activity. Since a labeled fragment corresponding to bp is generated, about 30 bp of fragments are generated regardless of the presence or absence of the cleavage activity of XPG, and thus the activity of XPG cannot be accurately analyzed. On the other hand, the use of a 3'-terminated bubbled substrate yields a label fragment having a size of about 30 bp or less only when cleavage by XPG occurs, so that a label fragment having a size of about 30 bp or less By confirming the amount of production of XPG activity can be quantitatively analyzed.

There is no restriction on the cells or tissues serving as samples for extracting the biological extract, and any cells or tissues requiring the activity analysis of XPG endonucleases can be used. For example, it may include all eukaryotic cells or tissues of mammals including humans. According to one embodiment of the present invention for analyzing XPG endonuclease activity, human fibroblasts or tissues of RKO cells or rats may be targeted, but are not limited thereto. Those skilled in the art can select and use appropriately.

As used herein, "biological extract" refers to an extract comprising a protein extracted from the nucleus, cells and / or tissues of an organism. Specifically, it may include cell nuclear extract, cell total protein extract, and / or tissue total protein extract.

As used herein, the term "cellular nuclear extract" means a content including all proteins in the nucleus extracted after the cytoplasm and the nuclear membrane of the eukaryotic cell is removed, and is used as distinguished from the purified protein. According to one embodiment, the nuclear extract may be obtained by treating cells with phosphatase inhibitors and protease inhibitors. However, the present invention is not limited thereto, and may be extracted using any extraction method known in the art.

As used herein, the term "cellular total protein extract" refers to extracting the contents including all proteins in cells by lysing eukaryotic cells, and is used as distinguished from purified proteins. According to one embodiment, the cell total protein extract may be obtained by treating the cells with a protease inhibitor. However, the present invention is not limited thereto, and may be extracted using any extraction method known in the art.

In the present specification, "tissue total protein extract" refers to extracting contents including all proteins in a specific tissue by dissolving a specific tissue (eg, liver, kidney, lung, stomach, etc.) of the organism. According to one embodiment, the tissue total protein extract may be obtained by placing the tissue in a lysis buffer and homogenizing, but the present invention is not limited thereto. Extraction can be made using any extraction method known in the art.

In the present specification, "DNA bubble substrate" in the DNA structure consisting of DNA double strands, both ends are complementary to form a double strand, but a part of the middle does not form a complementary bond to open the shape like bubbles (drops) It means the DNA structure formed. The bubble substrate acts as a substrate of XPG endonuclease as a bubble site where two strands of DNA are not paired is recognized as a damage site, and XPG recognizes the bubble structure of the bubble substrate to determine the 3 'end of the bubble structure. Will cut.

In one embodiment of the invention, the bubble substrate is SEQ ID NO: 1 (parent strand: 5'-CCA GTG ATC ACA TAC GCT TTG CTA GGA CAT CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CAG TGC CAC GTT GTA TGC CCA CGT TGA CCG-3 ') and SEQ ID NO: 2 (substrand: 5'-CGG TCA ACG TGG GCA TAC AAC GTG GCA CTG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT ATG TCC TAG CAA AGC GTA TGT GAT CAC TGG-3' Oligonucleotides) may be complementarily bound. The oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 are complementarily bonded to both ends to form a double strand, but complementary binding at the intermediate C repeat (SEQ ID NO: 1) and T repeat (SEQ ID NO: 2) site It does not form and opens and forms a bubble shape.

In another embodiment of the invention, the assay method according to the invention is characterized by the use of a DNA bubble substrate with a detectable label attached to the 3′-end. In this case, the detectable label may be a radioisotope, a fluorescent material, a light emitting material, or a precursor of the light emitting material. Specifically, ³² P, ³⁵ S, ¹³¹ I, ¹²³ I, ¹²⁵ I, ³ H, carboxyfluorescein (FAM), tetramethyltamine (TAMRA), Cy3, Cy5, IRDye series, fluorescein , Fluorescein isocyanate (FITC), Rhodamine, Texas Red, Alexa family, Dixygeninine (DIG) and biotin.

Attaching the detectable label to the DNA bubble substrate is well known in the art, and one of ordinary skill in the art can select and use an appropriate method from among known methods.

In another embodiment of the present invention, the mixing of the biological extract and the bubble substrate may be performed in the presence of a reaction buffer. In this case, the reaction buffer may be, for example, a HEPES buffer, but is not limited thereto.

In another embodiment of the present invention, the reaction buffer may have a pH of 6.0 to 8.5, specifically pH 6.0 to 8.0, pH 6.5 to 8.0, pH 6.5 to 7.5 or pH 6.5 to 7.0. This pH range is the range in which optimal binding to the DNA bubble substrate of XPG is possible.

According to another embodiment of the present invention, the method may further comprise adding MgCl ₂ or MnCl ₂ as a cofactor in the mixing step of the biological extract and the bubble substrate, wherein the MgCl ₂ or MnCl ₂ is the reaction It may be included in the buffer. The amount of MgCl ₂ or MnCl ₂ added may be 2-10 mM, which is an optimal binding range for XPG's DNA bubble substrate.

We performed a quantitative endonuclease activity assay of XPG activity derived from endogenous protein extracts. A "DNA bubble" with a detectable label (e.g., a radioisotope label or a non-radioactive label) at the 3'-end is an XPG-compatible substrate, which is a biological extract (cell nuclear protein extract, Cell total protein extracts and / or tissue total protein extracts) can be successfully used to cause structure specific DNA cleavage. In one embodiment of the present invention, nuclear protein extracts extracted from normal fibroblast and human colon cancer RKO cells were compared to XPG-deficient cells (XPG-null fibroblasts and XPG knockdown RKO cells, respectively). Compared to purified human XPG proteins, XPG cleavage analysis of tissue total protein extracts extracted from human and rat tissues (liver and kidney) has been shown to be successfully constructed. In addition, optimization was performed under different conditions for each sample type. The main advantage of the direct endogenous XPG based assay according to the present invention is that it is not labor intensive, economical and highly reproducible. The XPG assay of the present invention can be applied to most cell types and tissues of interest.

The present invention provides an effective assay method capable of quantitatively analyzing XPG endonuclease activity of cells or tissues. According to the present invention, the XPG endonuclease activity can be analyzed simply and inexpensively without the need for overexpression or purification of the recombinant protein, as well as the XPG endonucle of cells or tissues using a relatively small amount of DNA substrate. The activity of the clease can be analyzed close to the activity of the actual organism, and there is an advantage of reducing the analysis time. In addition, the kit for quantitative analysis of endonuclease activity of XPG according to the present invention may be used for clinical diagnosis for identifying DNA damage or for screening for therapeutic agents such as the oncogenic syndrome and skin cancer.

1 shows XPG activity analysis methods and results using 3'- and 5'-terminal labeled bubble nucleotide substrates. (A) is a schematic representation of an analytical method using 3'- and 5'-terminal labeled bubble substrates, with an asterisk (*) indicating a radiolabel. (B) shows the results of analysis using 3 'and 5' terminal labeled bubble substrates.
Figure 2 shows the results of testing the cleavage activity of intracellular cell nuclear XPG (A) time or (B) dose dependent.
3 shows the effect of important factors affecting XPG activity. (A) shows MgCl ₂ , (B) shows MnCl ₂ , and (C) shows the change in buffer pH.
4 shows the results of radioactive based quantitative XPG activity assays in various cell lines. (A) shows the result of Western blot analysis of the relative amount of XPG expressed from the nuclear extract of normal XPG cells and XPG-deficient cells. (B) shows the results of XPG cleavage activity analysis using nuclear extracts of normal XPG cells and XPG-deficient cells. Purified human XPG protein (+) hXPG was used as a positive control.
Figure 5 shows the results of radioactivity-based in vitro and in vivo XPG cleavage activity analysis. (A) shows the results of analyzing XPG cleavage activity using the cell total protein extract in normal XPG cells (RKO). (B) shows the results of analyzing XPG cleavage activity using a tissue total protein extract in rat tissue (liver).
Figure 6 shows the results of in vitro XPG cleavage activity analysis based on non-radioactive. (A) shows a comparison of serially diluted spot intensities of a sample in the 0-100% biotin range (bottom panel) and biotin oligo standard (top panel). (B) shows the results of analyzing XPG cleavage activity using nuclear protein extracts of normal XPG cells (RKO). (C) shows the results of analyzing the XPG cleavage activity using the cell total protein extract of normal XPG cells (RKO).

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example  1: Materials and Methods

1-1: Cell Culture

XPG shRNA (short-hairpin RNA) knockdown cells constructed using human colon cancer RKO cells (ATCC NO. CRL-257) were cultured in RPMI 1640 medium containing 10% FBS and 0.5 mg / ml puromycin, wild type RKO cells were cultured in RPMI 1640 medium to which (ATCC NO. CRL-257) 10% fetal calf serum (FBS) was added. Normal human fibroblasts (GM08399) and XPG null fibroblasts (GM16398) cells (Coriell Cell Repository, Camden, NJ, USA) were cultured in DMEM medium containing 10% FBS. The culture was performed at 37 ° C. and 5% CO ₂ .

Knockdown of XPG with shRNA vectors was performed as follows: Using the HiSpeed Plasmid Midi kit (Qiagen, Germany) in Escherichia coli clones with retroviral vector pSM2c (Open Biosystems, USA) containing XPG-specific shRNA targets The plasmid was extracted (plasmid extraction). Purified plasmids were sequenced to identify XPG-specific shRNA targets (sense: CCCACAGACTCAGTTCCAA, antisense: TTGGAACTGAGTCTGTGGG). XPG shRNA knockdown cells were prepared using the transfection reagent Fugen6 (Roche, Germany).

1-2: animals Care

Sprague-Dawley rats, 5 weeks old and about 200 g body weight, were purchased from Orient Bio Inc. (Seongnam, Korea). These animals were stored in plastic cages in a ventilated laboratory. With a 12 hour day / night cycle, the laboratory temperature was maintained at 20 + 2 ° C. and relative humidity 60 + 10%. Rats were given an ad libitum of water and laboratory complete food. These animals were domesticated for seven days before the experiment. All animal experiments were conducted according to the Animal and Ethics Review Committee of Sookmyung Women's University, Korea.

1-3: Cell Nucleus Extract ( cellular nuclear extract ) Ready

Nuclear extracts were prepared using a Caymans Nuclear Extraction kit (Caymanchem, USA). Specifically, 1 × 10 ⁶ RKO cells and fibroblasts were planted in a 100-mm culture dish. After centrifugation the cell pellets were washed twice with 5 ml of ice cold PBS in the presence of phosphatase inhibitors and centrifuged again. The pellets were resuspended in 500 μl of chilled (1 ×) hypotonic buffer and incubated for 15 minutes on ice. 50 μl of (10%) Nonidet P-40 was added to dissolve the cell membrane, followed by simple centrifugation for 14000 g for 30 seconds. The pelleted nuclei was lysed after lysis with 50 μl of chilled (1 ×) complete nuclear extraction buffer containing a mixture of protease and phosphatase inhibitors. After centrifugation, the cell supernatant was collected and stored at -80 ° C. This protein concentrate was then quantified.

1-4: Cell Total protein  Extract ( cellular total protein extract ) Ready

1x106 cells of 10-ml suspension were each planted in a 100-mm petri dish, and after incubation, the cells were scraped and centrifuged at 1500 rpm for 5 minutes to sink the cells. The cells were then subjected to 100 μl RIPA Lysis buffer [50 mM Tris-HCl (pH 7.5), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1% (v / v) Triton- for 30 minutes. 100, 0.1% (v / v) sodium dodecyl sulfate (SDS), 1 mM dithiothreitol (DTT) and protease inhibitor cocktail]. Total cell lysates were obtained by centrifugation at 13,000 rpm for 30 minutes and then stored at -80 ° C. This protein was quantitated.

1-5: Organization Total protein  Extract ( tissue total protein extract ) Ready

10-40 mg of rat liver and kidney tissue were placed in 500 μl of Lysis buffer PRO-PREP (Intron Biotechnology, Republic of Korea) and chopped with surgical scissors in an ice bath. Finely chopped tissues were homogenized and vortexed and incubated on ice for 30 minutes. The supernatant was collected and centrifuged at 13,000 rpm for 15 minutes and the samples were divided and stored at -80 ° C. This protein concentrate was finally quantified.

1-6: Western Blot  Cell nucleus by analysis XPG  Confirm

The nuclear extract was separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked. XPG protein was detected using mouse monoclonal antibodies against XPG and peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, USA) as secondary antibodies. Then chemiluminescence was performed (ECL Plus Western blotting detection kit, GE Healthcare, UK). CyclinB1 was used as a protein loading control. The whole procedure was performed three times each.

1-7: Analytical Methods Based on Radioactive Systems

3 'end is a radioisotope Labeled DNA bubble  Temperament Preparation

Bubble oligonucleotide substrates were used as specific substrates for XPG cleavage activity analysis. One strand was first 3'-terminated with terminal transferase (NEB, England) and [a- ³² P] dATP as follows. The first DNA strand, 20 pmol of bubble-up oligonucleotide (SEQ ID NO: 1) was labeled with (1X) terminal transferase buffer, 250 mM CoCl ₂ , 100 mM [α- ³² P] dATP and 10 units of terminal transferase. To the reaction mixture (50 μl) was added. After incubation at 37 ° C. for 30 minutes, it was heated to 70 ° C. for 10 minutes to inactivate it. The final labeled bubble-up oligonucleotides were precipitated with 1 μl glycogen and 36 μl isopropanol and then centrifuged at 15,000 rpm for 10 minutes at room temperature. The water bath was then added with 38 μl (1 ×) of annealing buffer (10 mM Tris pH 8.0, 1 mM EDTA and 100 mM NaCl) and the same amount of unlabeled second DNA strand, bubbledown oligonucleotide (SEQ ID NO: 2). The annealing step was performed by cooling from 95 ° C. at.

5 'end to radioisotope Labeled DNA bubble  Temperament Preparation

The 5'-terminal radioisotope labeling procedure of the bubble substrate was performed almost identically to the 3'-terminal radioisotope procedure as mentioned in Examples 1-6 above. The first bubbledown oligonucleotide strand was first 5'-labeled with T4 polynucleotide kinase (Promega, USA) and [γ- ³² P] ATP. 20 pmol of a second bubbleup oligonucleotide was then added to the labeling reaction mixture (50 μl) containing (1 ×) T4 polynucleotide kinase buffer, [γ- ³² P] ATP and 20 units of T4 polynucleotide kinase. The subsequent procedure was the same as the 3'-terminal radioisotope labeling procedure.

With radioactive isotopes Labeled DNA Marker  Ready

Radiolabeled 5'-ends of DNA markers were performed using [γ- ³² P] ATP and T4 polynucleotide kinases. The DNA size marker used was dephosphorylated Φ × 174 DNA / HinfI (Promega, USA). Basically 50-200 ng of DNA marker was mixed with 10 μl of reaction mixture [(1 ×) T4 polynucleotide kinase buffer, 2 mM [γ- ³² P] ATP and 10 units of T4 polynucleotide kinase] and then at 37 ° C. Incubate for 10 minutes. The reaction was stopped by addition of the same volume of (1 ×) sample loading buffer [90% formamide, 5 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol]. Samples were heated at 95 ° C. for 3 minutes before gel electrophoresis was performed.

Radioactive Base Using Nuclear Cell Extracts XPG  Cleavage analysis

XPG endonuclease assays are described by E. Evans et al. (E. Evans, J. Fellow, A. Coffer, and RD Wood, EMBO. 16 (1997) 625-638) and Y. Habraken et al. Based on the in vitro analytical procedures described in A. Constantinou, D. Gunz, E. Evans, P. Lalle, PA Bates, RD Wood, and SG Glarkson, J. Biol. Chem. 274 (1999) 5637-5648. This was done with some modification. Nucleate extract (typically 50-100 ng) and 1 pmol of radioisotope labeled bubble oligonucleotide substrate were added to 8 μl of reaction buffer [(25 mM HEPES pH 6.8, 10% glycerol, 2 mM MgCl ₂ , 50 mg / ml bovine serum albumin (BSA) and 1 mM DTT] were mixed for 30 minutes at 37 ° C. The reaction was stopped by adding an equal volume of (1 ×) sample loading buffer The sample was heated at 95 ° C. for 3 minutes and then 12 Separation by% SDS-PAGE (1 hour at 1,000 V.) The gel was then transferred to Whatman® 3-mm paper, dried and visualized by autoradiography All analyzes were performed twice each.

cell Total protein  Radioactive Base Using Extracts XPG  Cleavage analysis

XPG endonuclease assays are described by E. Evans et al. (E. Evans, J. Fellow, A. Coffer, and RD Wood, EMBO. 16 (1997) 625-638) and Y. Habraken et al. (A. Constantinou, D. Gunz, E. Evans, P. Lalle, PA Bates, RD Wood, and SG Glarkson, J. Biol. Chem. 274 (1999) 5637-5648) disclosed in Based on the in vitro assay procedure, some modifications were performed. 8 μl of reaction buffer [(25 mM HEPES pH 6.8, 10% glycerol, 2 mM MgCl ₂ ,) with cell total protein extract (typically using 50-100 ng protein) and 1 pmol of radioisotope labeled bubble oligonucleotide substrate. 50 mg / ml bovine serum albumin (BSA) and 1 mM DTT] were mixed for 30 minutes at 37 ° C. The reaction was stopped by adding an equal volume of (1 ×) sample loading buffer The sample was heated at 95 ° C. for 3 minutes. Then separated by 12% SDS-PAGE (1 hour at 1,000 V.) The gels were then transferred to Whatman® 3-mm paper, dried and visualized by autoradiography, all analyzes were performed twice each.

group Total protein  Radioactive Base Using Extracts XPG  Cleavage analysis

XPG endonuclease assays are described by E. Evans et al. (E. Evans, J. Fellow, A. Coffer, and RD Wood, EMBO. 16 (1997) 625-638) and Y. Habraken et al. (A. Constantinou, D. Gunz, E. Evans, P. Lalle, PA Bates, RD Wood, and SG Glarkson, J. Biol. Chem. 274 (1999) 5637-5648) disclosed in vitro This was done with some modifications based on the analytical procedure. Tissue total protein extract (typically using 500-1000 ng protein) and 1 pmol of radioisotope labeled bubble oligonucleotide substrate were added to 8 μl of reaction buffer [(25 mM HEPES pH 6.8, 10% glycerol, 2 mM MgCl ₂ , 50 mg / ml bovine serum albumin (BSA) and 1 mM DTT] were mixed for 30 minutes at 37 ° C. The reaction was stopped by adding an equal volume of (1 ×) sample loading buffer The sample was heated at 95 ° C. for 3 minutes. Then separated by 12% SDS-PAGE (1 hour at 1,000 V.) The gels were then transferred to Whatman® 3-mm paper, dried and visualized by autoradiography, all analyzes were performed twice each.

1-8: Non-radioactive  System based analysis method

3'-end By biotin Labeled DNA bubble  Temperament Preparation

Examples 1-7 describe methods when using radioisotopes as detectable labels attached to the 3'-end of a DNA bubble. In this embodiment, a method of using a label other than a radioisotope will be described. Bubble oligonucleotide substrates were used as specific substrates for XPG cleavage activity analysis. One strand was first 3'-terminated with a terminal transferase (Thermo Scientific, USA) and biotin-11-UTP as follows to make a biotinylated 3'-end. The first DNA strand, 5 pmol of bubble-up oligonucleotide (SEQ ID NO: 1), was loaded with (1X) terminal transferase buffer, 0.5 μM biotin-11-UTP, and one unit of terminal transferase (Thermo Scientific, USA). To a labeled reaction mixture containing 50 μl. Incubate at 37 ° C. for 30 minutes. The final labeled bubble-up oligonucleotides were precipitated with 1 μl glycogen and 36 μl isopropanol and then centrifuged at 15,000 rpm for 10 minutes at room temperature. The water bath was then added with 38 μl (1 ×) of annealing buffer (10 mM Tris pH 8.0, 1 mM EDTA and 100 mM NaCl) and the same amount of unlabeled second DNA strand, bubbledown oligonucleotide (SEQ ID NO: 2). The annealing step was performed by cooling from 60 ° C. at. Samples were stored at -20 ° C until use.

By biotin Labeled DNA Marker  Ready

The biotin labeling of the 5'-end of the DNA marker was performed using biotin-11-UTP and T4 polynucleotide kinase. The DNA size marker used was dephosphorylated Φ × 174 DNA / HinfI (Promega, USA). Basically, 50-200 ng of DNA marker is mixed with 10 μl of reaction mixture [(1 ×) T4 polynucleotide kinase buffer, 0.5 μM biotin-11-UTP and 10 units of T4 polynucleotide kinase] and then at 37 ° C. for 10 minutes. Incubated. The reaction was stopped by addition of the same volume of (1 ×) sample loading buffer [90% formamide, 5 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol]. Samples were heated at 95 ° C. for 3 minutes before gel electrophoresis was performed. Samples were stored at -20 ° C until use.

Using nucleus extract Non-radioactive  base XPG  Cleavage analysis

XPG endonuclease assays are described by E. Evans et al. (E. Evans, J. Fellow, A. Coffer, and RD Wood, EMBO. 16 (1997) 625-638) and Y. Habraken et al. (A. Constantinou, D. Gunz, E. Evans, P. Lalle, PA Bates, RD Wood, and SG Glarkson, J. Biol. Chem. 274 (1999) 5637-5648) disclosed in vitro This was done with some modifications based on the analytical procedure. Nucleate extract (typically 50-100 ng) and 1 pmol of biotin at the 3'-terminus of the bubble oligonucleotide substrate were added to 8 μl of reaction buffer [(25 mM HEPES pH 6.8, 10% glycerol, 2 mM MgCl ₂ , 50 mg / ml bovine serum albumin (BSA) and 1 mM DTT] for 30 minutes at 37 ° C. The reaction was stopped by adding an equal volume of (1 ×) sample loading buffer. (3 hours at 100 V.) The gel was then electrophoresed to a positively charged nylon membrane at 25 V / 250 mA for 1 hour and fixed using UVC crosslinking at 0.120 J / cm ² for 1 minute. Detected by chemiluminescent signal using Chemilunescent Nucleic Acid Detection Module (Thermo Scientific, USA) In brief, blotted membranes are blocked by gently shaking for 15 minutes with an initially preheated (37-50 ° C) blocking buffer. And then stabilized streptabi Incubation was with gentle shaking for 15 minutes with a conjugate / blocking buffer containing Dean-horseradish peroxidase conjugate (1: 300 dilution) The membranes were washed four times for 5 minutes each with 1 × wash buffer. Previously the membrane was equilibrated with the substrate equilibration buffer with gentle shaking, after which the membrane was carefully removed from the substrate equilibration buffer to contain a clean container containing the substrate working solution (luminol / enhancer solution: 1: 1 mixing ratio of stable peroxide solution). The membrane was removed from the substrate working solution by blotting the edges of the membrane with paper towels to remove excess buffer and 2-5 minutes on an X-ray film (according to the desired signal). Were exposed and the film was developed All analyzes were performed twice each.

cell Total protein  With extract Non-radioactive  base XPG  Cleavage analysis

XPG endonuclease assays are described by E. Evans et al. (E. Evans, J. Fellow, A. Coffer, and RD Wood, EMBO. 16 (1997) 625-638) and Y. Habraken et al. (A. Constantinou, D. Gunz, E. Evans, P. Lalle, PA Bates, RD Wood, and SG Glarkson, J. Biol. Chem. 274 (1999) 5637-5648) disclosed in Based on the in vitro assay procedure, some modifications were performed. 8 μl of reaction buffer [(25 mM HEPES pH 6.8, 10% glycerol, 2) was added to the cell total protein extract (typically using 50-100 ng protein) and 1 pmol of the biotin-labeled bubble oligonucleotide substrate at 3'-terminus. mM MgCl ₂ , 50 mg / ml bovine serum albumin (BSA) and 1 mM DTT] for 30 minutes at 37 ° C. The reaction was stopped by adding an equal volume of (1 ×) sample loading buffer. -PAGE (3 hours at 100 V.) The gel was then electrophoresed to a positively charged nylon membrane at 25 V / 250 mA for 1 hour and fixed using UVC crosslinking at 0.120 J / cm ² for 1 minute. The membranes were detected by chemiluminescent signals using a Chemilunescent Nucleic Acid Detection Module (Thermo Scientific, USA) All analyzes were performed twice each.

group Total protein  With extract Non-radioactive  base XPG  Cleavage analysis

XPG endonuclease assays are described by E. Evans et al. (E. Evans, J. Fellow, A. Coffer, and RD Wood, EMBO. 16 (1997) 625-638) and Y. Habraken et al. (A. Constantinou, D. Gunz, E. Evans, P. Lalle, PA Bates, RD Wood, and SG Glarkson, J. Biol. Chem. 274 (1999) 5637-5648) disclosed in Based on the in vitro assay procedure, some modifications were performed. Tissue total protein extract (typically 500-1000 ng protein) and 1 pmol of 3'-terminus of biotin-labeled bubble oligonucleotide substrate were immobilized in 8 μl of reaction buffer [(25 mM HEPES pH 6.8, 10% glycerol, 2 mM MgCl ₂ , 50 mg / ml bovine serum albumin (BSA) and 1 mM DTT] for 30 minutes at 37 ° C. The reaction was stopped by addition of an equal volume of (1 ×) sample loading buffer. SDS-PAGE (3 h at 100 V) The gel was then electrophoresed to a positively charged nylon membrane at 25 V / 250 mA for 1 h and fixed using UVC crosslinking at 0.120 J / cm ² for 1 min. The membranes were detected by chemiluminescent signals using a Chemilunescent Nucleic Acid Detection Module (Thermo Scientific, USA) All analyzes were performed twice each.

Example  2: XPG  Activity analysis

Conventionally, XPG cleavage activity assay had to use XPG purified. In particular, the ERCC1-XPF complex cleaves the 5'-terminus of the substrate, thus making the conventional XPG activity assay poorly done (FIG. 1A). To overcome this problem, the present invention provides a method for analyzing XPG cleavage activity by labeling the 3′-end of a substrate with a detectable label (FIG. 1B).

The left panel of Figure 1B shows the results with a 5'-end labeled bubble substrate and the right panel with a 3'-end labeled bubble substrate. C is a negative control without a nuclear extract, M represents a DNA marker, lanes 2 and 3 are the results of 10 and 30 minutes of reaction using a 5 'terminal labeled substrate, lanes 5 and 6, respectively. Shows the result of 10 and 30 minutes of reaction using a 3'-terminal labeled substrate.

As shown in FIG. 1B, when the 5'-terminal labeled substrate was used, no nucleotide fragment of about 60 bp that could confirm the cleavage activity of XPG was observed, whereas the ERCC1-XPF complex having 5 'terminal cleavage activity was not shown. The effect confirmed that only nucleotide fragments smaller than 30 bp were produced (lanes 1-3).

However, with the 3'-terminal labeled substrate, about 30 bp of cleavage activity of XPG was observed in the presence of nuclear extracts (lanes 5-6) compared to the absence of nuclear extracts (lanes 4). It was confirmed that much more nucleotide fragments were produced.

These results indicate that the 3'-end labeled bubble substrate is a suitable substrate for efficient XPG activity analysis.

Example  3: time or dose dependent XPG  Activity analysis

Based on the previous results, the cleavage activity of XPG on bubble substrates labeled with 3'-terminus radioisotopes was time- or dose-dependently analyzed. Unless stated otherwise, incubation of a mixture of 3'-end labeled bubble substrate and nuclear extract under standard XPG assay conditions was performed time dependent. XPG of nuclear extracts can specifically cleave substrates. In FIG. 2A, C represents a control group without nuclear extract, M represents a DNA marker, and lanes 2 or 5 are incubated for 10 minutes, and lanes 3 or 6 are incubated for 30 minutes. In the presence of XPG nuclear extract, the cleaved products showed a constant migration rate of about 30 bp. Longer incubation time increased the amount of nucleotide cleavage product, which was inversely proportional to the amount of substrate that was not cleaved. These results show that the cleavage of DNA bubble substrate by XPG nuclear extract is time-dependent. In parallel experiments, the dependence of XPG endonuclease activity on the concentration of the extract was examined. In FIG. 2B, C represents a negative control without nuclear extract, M represents a DNA marker, and lanes 2 or 4 show 50 ng, and lanes 3 or 5 show 100 ng of nuclear extract. Experimental results confirmed that the cleavage activity of XPG is proportional to the amount of nuclear extract.

As a result, it can be seen that XPG endonuclease activity assay using nuclear extract appears time and dose dependent.

Example  4: XPG  Identification of Factors Influencing Activity

The effect of different parameters (divalent cofactor and pH of reaction buffer) on the cleavage efficiency of XPG was examined. After reconstructing the XPG cleavage reaction for 30 minutes of 100 pmol 3'-terminal radioisotope labeled bubble substrates and 100 ng of nuclear extracts in 37 ° C. reaction buffer under various individual factors, DNA banding patterns and Intensity was measured.

First incubation was carried out by mixing the nucleus extract with a bubble substrate labeled 3'-terminally radioisotope in the presence of MgCl ₂ or MnCl ₂ (2-10 mM). 3A is a result showing the effect of MgCl ₂ , C represents a negative control, M represents a DNA marker, lane 2 is 2 mM, lane 3 is 5 mM, lane 4 is 7 mM, lane 5 is 10 mM of MgCl2 The case of addition is shown. Increasing the MgCl ₂ concentration showed improved XPG reaction efficiency, with 7 mM MgCl _{2 showing} the most efficient optimal XPG response. 3B shows the effect of MnCl ₂ , where C represents a negative control, M represents a DNA marker, lane 2 is 2 mM, lane 3 is 5 mM, lane 4 is 7 mM, lane 5 is 10 mM of MnCl ₂ The case where it adds is shown. Likewise, as the MnCl ₂ concentration was increased, improved XPG endonuclease activity was shown.

To determine the effect of pH of the reaction buffer on the XPG cleavage efficiency of the nuclear extract, experiment was performed with HEPES buffer of pH 6.8 or pH 7.9. In FIG. 3C, C represents a negative control, M represents a DNA marker, and both buffer pH 7.9 and buffer pH 6.8 showed XPG endonuclease activity, but XPG endonuclease activity at buffer pH 6.8 was higher than buffer pH 7.9. High.

Example  5: XPG  Normal cells XPG Radioactive base in deficient cells XPG  Active comparison

Quantitative analysis of XPG endonuclease activity in human fibroblasts and human colon cancer RKO cells was performed against XPG-deficient cells.

4A shows the results of Western blot analysis of the relative amount of XPG protein in nuclear extracts extracted from normal XPG cells (fibroblasts and RKO) and XPG-deficient cells (XPG-free fibroblasts and XPG shRNA knockdown RKO cells). . CyclinB1 is a loading control. In the nuclear extract derived from XPG-deficient cells, the expression level of XPG was confirmed to be significantly reduced compared to the nuclear extract derived from normal cells.

FIG. 4B shows the results of incubation of XPG nuclear extracts from various cells and bubble substrates labeled with radioisotopes at 3 ′ ends in a reaction buffer of pH 6.8 containing 7 mM MgCl ₂ , where C is a negative control, M is a DNA marker and (+) hXPG is a purified human XPG protein, indicating a positive control. It was confirmed that relatively high XPG-mediated DNA cleavage occurred in normal fibroblasts (lane 2) compared to cells without XPG (lane 3). Similarly, XPG shRNA knockdown cells (lane 5) were relatively higher in RKO cells (lane 4) than in XPG DNA cleavage activity. DNA cleavage activity of the XPG nuclear extract of the present invention can be seen that the reproducibility is high compared to the activity of purified recombinant human XPG (Fig. 4B, lanes 6 and 7) as a positive control. Through these results, it is possible to confirm the optimized XPG activity analysis conditions of the present invention.

Example  6: in vitro  And in vivo Radioactive base XPG  activation

XPG Activity Analysis in Nuclear Extracts Following XPG endonuclease activity assays were performed using total protein extracts from human cells and rat tissues. Purified human XPG protein was used as a positive control.

Figure 5A shows the results of analyzing the XPG cleavage activity using the cell total protein extract in normal XPG cells (RKO), the purified human XPG protein (+) hXPG as a positive control was compared. XPG activity on a bubble substrate (1 pmol) labeled 3'-terminally with radioisotopes varies with the amount of cell total protein extract and the incubation time. In 37 ° C. reaction buffer, lane 2 is 50 ng, lanes 3 and 4 are 100 ng, lanes 5 are 50 ng, lanes 6 are 100 ng, lanes 2 and 3 are 10 minutes, lanes 4 are 30 minutes, lanes 5 and 6 Shows the result at 30 minutes. C represents negative control and M represents DNA marker. Figure 5B shows the results of analyzing XPG cleavage activity using tissue total protein extract in rat tissue (liver), the purified human XPG protein (+) hXPG as a positive control was compared. XPG activity on a bubble substrate (1 pmol) labeled 3'-terminally with radioisotopes varies with the amount of cell total protein extract and the incubation time. In 37 ° C reaction buffer, lane 2 is 500 ng, lanes 3 and 4 are 1000 ng, lanes 5 are 50 ng, lanes 6 are 100 ng, lanes 2 and 3 are 10 minutes, lanes 4 are 30 minutes, lanes 5 and 6 Shows the result at 30 minutes. C represents negative control and M represents DNA marker.

Incubation of XPG total extract (50-100 ng of cell total protein extract) derived from human cells for 10 to 30 minutes in pH 6.8 reaction buffer with 7 mM MgCl ₂ results in cleavage products of ˜30 nucleotides. It is time and dose dependent as shown in lane 2 in 3 and lane 3 in FIG. 5A. Similarly, incubating XPG total extract (500-1000 ng tissue total protein extract) derived from rat tissue for 10 to 30 minutes in pH 6.8 reaction buffer containing 7 mM MgCl ₂ resulted in a cleavage product of ˜30 nucleotides. Occurs. It is time and dose dependent as shown in lanes 2 to 3 and 4 in lanes 3 of FIG. 5B. The DNA pattern generated from the total protein extract of human cells or tissue cells is consistent with the DNA pattern of the human XPG protein (lanes 5 and 6 of FIGS. 5A and 5B), in which the cleavage product is an endonuclease of XPG. By activity. These results in vitro and in It is shown that XPG activity assay of the present invention using total protein extracts in both vivo was successfully performed.

Example  6: in vitro in Non-radioactive  base XPG  activation

Radioisotopes are expensive and dangerous and have short lifespans. Thus, as an alternative, the 3'-end of the DNA bubble substrate was labeled with biotin. The 3'-end of the bubble substrate was labeled with biotin by the method described in Examples 1-8. 1-3 biotinylated ribonucleotides were added to the 3'-terminus of the bubble-shaped oligonucleotides in the presence of Co ²⁺ using terminal deoxynucleotidyl transferase. Combined. Biotin-mediated tailing of the bubble oligonucleotides then took place. Labeling efficiency was performed by dot blotting and spot intensity of the spot samples was indicated by chemiluminescence using a streptavidin-horseradish peroxidase (HRP) conjugate detection system and compared with the biotin control oligo standard (0-100% biotin). .

6A shows a comparison of serially diluted spot intensities of a sample in the 0-100% biotin range (bottom panel) and biotin oligo standard (top panel). As shown in the upper and lower panels of FIG. 6A, the biotin labeling efficacy of the samples can be compared to the control standard. FIG. 6B shows the results of analyzing XPG cleavage activity using nuclear protein extracts of normal XPG cells (RKO). Purified human XPG protein (+) hXPG was used as a positive control. XPG activity on the bubble substrate (1 pmol) labeled 3′-end with biotin varies with the amount of nuclear extract and incubation time. In 37 ° C. reaction buffer, lane 2 is 50 ng, lanes 3 and 4 are 100 ng, lanes 5 are 50 ng, lanes 6 are 100 ng, lanes 2 and 3 are 10 minutes, lanes 4 are 30 minutes, lanes 5 and 6 Shows the result at 30 minutes. C represents negative control and M represents DNA marker. Figure 6C shows the results of analyzing the XPG cleavage activity using the cell total protein extract of normal XPG cells (RKO), the purified human XPG protein (+) hXPG as a positive control was compared. XPG activity on the bubble substrate (1 pmol) labeled 3′-end with biotin varies with the amount of nuclear extract and incubation time. In 37 ° C. reaction buffer, lane 2 is 50 ng, lanes 3 and 4 are 100 ng, lanes 5 are 50 ng, lanes 6 are 100 ng, lanes 2 and 3 are 10 minutes, lanes 4 are 30 minutes, lanes 5 and 6 Shows the result at 30 minutes. C represents negative control and M represents DNA marker.

Incubation of nuclear extracts (50-100 ng of protein extract) of human cells for 10-30 minutes in pH 6.8 reaction buffer containing 7 mM MgCl ₂ results in cleavage products of ˜30 nucleotides. It is time and dose dependent as shown in lane 2 in 3 and lane 3 in FIG. 6B and corresponds to the cleavage product of positive XPG shown in lanes 5 and 6 in FIG. 6B. Similarly, incubating the cell extract (50-100 ng of cell total protein extract) for 10 to 30 minutes in a pH 6.8 reaction buffer containing 7 mM MgCl _{2 results} in the cleavage product of ˜30 nucleotides. It is time and dose dependent as shown in lanes 2 to 3 and 4 in lanes 6 of FIG. 6C and corresponds to the cleavage products of positive XPG shown in lanes 5 and 6 of FIG. 6C.

This indicates that the cleavage product of the DNA bubble substrate is due to the endonuclease activity of XPG. In addition, it can be seen that the use of a non-radioactive label such as biotin shows the same results as the radiolabel.

As discussed above, the XPG activity assay of the present invention has the following advantages: 1) no hassle of overexpression and purification to produce recombinant protein, 2) low cost, 3) application It is an easy-to-use assay, 4) applicable to various cell and tissue tests, 5) low amount of DNA substrate used, and 6) short analysis time. XPG activity assays are very important in clinical diagnostics, drug development and cancer treatment. The XPG activity assay of the present invention is useful for evaluating XPG activity against toxicity of the surrounding environment, such as UV radiation or chemical mutagens. In addition, the XPG activity assay of the present invention can be performed using XPG derived from various biological extracts while maintaining the reproducibility and sensitivity of the assay. Thus, the assays of the present invention can be used to simultaneously assess XPG activity of a cell or tissue of interest.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (12)

Preparing a biological extract sample containing xeroderma pigmentosum (XP) of the complementation group G;
Preparing a DNA bubble substrate;
Attaching a detectable label to the 3 'end of the DNA bubble substrate to form a DNA bubble substrate labeled with the 3'end; And
Mixing the biological extract sample with the 3 'end labeled DNA bubble substrate;
XPG endonuclease activity analysis method comprising the step.
The method of claim 1,
The biological extract sample is a cell nuclear extract, cell total protein extract or tissue total protein extract XPG endonuclease activity analysis method.
The method of claim 1,
The DNA bubble substrate is an XPG endonuclease activity analysis method in which the oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 are complementary bound. The method of claim 1,
XPG endonuclease activity assay method wherein the detectable label is a radioisotope, fluorescent material, luminescent material or precursor of luminescent material.
5. The method of claim 4,
The detectable label is ³² P, ³⁵ S, ¹³¹ I, ¹²³ I, ¹²⁵ I, ³ H, carboxyfluorescein (FAM), tetramethyltamine (TAMRA), Cy3, Cy5, IRDye series, fluorescein ( XPG selected from the group consisting of fluorescein, fluorescein isocyanate (FITC), rhodamine, Texas red, Alexa family, dioxygeninine (DIG) and biotin Endonuclease Activity Assay Method.
The method of claim 1,
The method further comprises the step of confirming that the DNA bubble substrate is cleaved after the step of mixing the biological extract sample and the 3 'terminal labeled DNA bubble substrate, XPG endonuclease activity analysis method.
The method according to claim 6,
The method is an XPG endonuclease activity assay method wherein the size of the cleaved DNA bubble is less than 30 nucleotides.
The method of claim 1,
XPG endonuclease activity assay method of mixing the biological extract sample and the DNA bubble substrate is carried out in the presence of a reaction buffer.
9. The method of claim 8,
The reaction buffer is XPG endonuclease activity assay method having a pH of 6.0 to 8.5.
9. The method of claim 8,
Wherein the reaction buffer comprises MgCl ₂ or MnCl ₂ XPG endonuclease activity analysis method.
11. The method of claim 10,
The amount of MgCl ₂ or MnCl ₂ is 2-10 mM XPG endonuclease activity analysis method.
XPG endonuclease activity assay kit comprising a biological extract according to claim 1 and a DNA bubble substrate.

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