WO2001032857A1 - Methylation of unstable sequences - Google Patents

Abstract

The present invention enables the modulation of the genetic stability of a nucleotide sequence.

Description

METHYLATION OF UNSTABLE SEQUENCES

Field of the Invention The invention relates to a method for modulating the genetic stability of nucleotide sequences.

Background of the Invention

Sites of DNA methylation are 'hotspots' for point mutations. Methylation at the 5 position of cytosine residues is known to cause C→T transition mutations via the spontaneous deamination of 5-methylcytosine to thymine (Coulondre et al., 1978), or through a methyltransferase-mediated process (Shen et al., 1992). Other forms of mutation involving large regions of DNA are also associated with altered methylation; for example the somatic instability of the unmethylated expanded (CGG)_n repeat of fragile X mental retardation (FRAXA) (Reyniers et al., 1993; Malther et al., 1997; Wohrle et al., 1993; Kambouris et al., 1996; Lachiewicz et al., 1996); the hypermethylation associated with certain cancer-associated deletions (Vachtenheim et al., 1994; Makos et al., 1993b; 1993b); the hypomethylation of satellites II and III, alpha-satellites and Alu repeats, associated with increased chromosomal instability found in patients with ICF Immunodeficiency, Centromeric instability and Facial abnormalities) (Jeanpierre et al., 1993); the mutagenic affects of the demethylating drug 5-azadeoxycytidine (Haaf, 1995); and the increased rates of mutation in mice lacking the major DNA methyltransferase, Dnmtl (Chen et al., 1998). Furthermore, certain tumors display a methylator phenotype (altered regulation of methylation) as well as a mutator phenotype, manifest as genome-wide microsatellite instabilities (Ahuja et al., 1997; Toyota et al., 1999). Together these findings suggest that there is a close relationship between methylation regulation and genome instability. However, aside from C→T transitions, the relationship of CpG methylation of any particular sequence to instability is not known. In June 2000, the completion of the first rough draft of the human genome was announced by the Human Genome Project and Celera Genomics. Many portions of the genome remain to be sequenced and in some cases, cloning and sequencing has been hampered by instability of portions of the sequence, for example where there are repetitive motifs. There is a need for methods to facilitate the cloning of unstable nucleotide sequences, so that such sequences can be successfully sequenced.

Summary of the Invention In accordance with one embodiment of the present invention, a method of modulating the genetic stability of a selected nucleotide sequence, comprises the steps of:

(a) providing a nucleotide sequence comprising the selected nucleotide sequence; (b) replicating the nucleotide sequence in a host cell capable of methylating the selected nucleotide sequence or a portion of the nucleotide sequence flanking the selected nucleotide sequence, thereby modulating the genetic stability of the selected nucleotide sequence. In accordance with another embodiment of the present invention, a method of enhancing the genetic stability of a selected unstable nucleotide sequence, comprises the steps of:

(a) providing a nucleotide sequence comprising the selected unstable nucleotide sequence; (b) replicating the nucleotide sequence in a host cell capable of methylating the selected unstable nucleotide sequence or a portion of the nucleotide sequence flanking the selected unstable nucleotide sequence, thereby enhancing the genetic stability of the selected unstable nucleotide sequence. In accordance with another embodiment of the present invention, a method for mutagenising a nucleotide sequence, comprises the steps of:

(a) providing a nucleotide sequence;

(b) replicating the nucleotide sequence in a host cell capable of methylating the nucleotide sequence, thereby mutagenising the nucleotide sequence.

In accordance with another embodiment of the present invention, a method for methylating a nucleotide sequence, comprises replicating the nucleotide sequence in a host cell capable of methylating the nucleotide sequence.

Summary of the Drawings

The present invention will be further understood from the following description with reference to the Figures, in which: Figure 1 shows the effect of methylation on the genetic stability of

FRAXA (CGG)n repeats. Panel A is a photograph of a gel showing DNA fragment lengths after restriction digestion of FRAXA plasmids containing (CGG)53 repeats and propagated in E. col] in the presence (+) or absence (-) of the Sss1 CpG methylase-expressing plasmid pAIT2, analysed by polyacryiamide gel electrophoresis and ethidium bromide staining. Panel B is an autoradiograph of the gel of Panel A probed with radiolabelled (CGG)ι₀ oligonucleotides. Panel C shows repeat length stability determined by densitometric analysis. Open bars indicate % material with starting length repeats and shaded bars indicate % deletion products. Panel D shows the magnitude of repeat loss assessed by culturing, colony isolation and DNA analysis. The % colonies with a given deletion length is shown after first (open bars), second (solid bars) or third (cross-hatched bars) sub-culturings. Panel E is a photograph of a gel, showing DNA fragment lengths from FRAXA plasmids, labeled as in Panel A. Figure 2 shows the effect of methylation on the genetic stability of DM (CTG)n repeats, analysed by restriction digestion and PAGE, as described for Figure 1. Panel A shows the results of 3 sub-culturings of a DM plasmid containing (CTG)83 repeats in the presence (+) or absence (-) of pAIT2. Panel B is an autoradiograph of the gel of Panel A, as described for Figure 1. Panel C is similar to Panel A for plasmids containing (CTG)30, (CTG)50, (CTG)83 and (CTG)100 repeats. Panel D shows (CTG)100 repeat plasmids cloned in stable ((CTG)100) and unstable ((CTG)100-) orientations.

Figure 3 shows the effect of methylation on the genetic stability of dinucleotide repeats, analysed by restriction digestion and PAGE, as described in Figure 1. Plasmids contained dinucleotides (TC)37, (CA)30 and (GC)13 in both cloning orientations.

Figure 4 shows the effect of methylation on the genetic stability of minisatellite and satellite 3 repeats, analysed by restriction digestion and agarose gel electrophoresis. Starting lengths are indicated by dark arrowheads, vector restriction fragments by light arrowheads and faster migrating DNA's, the products of deletion events, by arrows.

Figure 5 shows in diagramatic form the components of plasmid pFXA53-SVB.

Detailed Description of the Invention Definitions

"Genetic stability" of a nucleotide sequence, as used herein, refers to its ability to maintain its nucleotide sequence without mutation.

"Modulate", as used herein, refers to a change in the genetic stability of a nucleotide sequence. The change may be an increase or a decrease in the genetic stability. "Unstable nucleotide sequence", as used herein, refers to a nucleotide sequence which is susceptible to genetic mutation.

"Enhancing the genetic stability of an unstable nucleotide sequence" as used herein, refers to enhancing the ability of the sequence to maintain its nucleotide sequence without mutation.

"Transforming", as used herein, refers to introducing an exogenous nucleotide into a host cell. Any known method for introducing an exogenous nucleotide sequence into a host cell may be employed. The method selected will depend on the type of host cell, as is known to those of skill in the art.

"Co-transforming" a host cell with an unstable nucleotide sequence and a methylase-encoding nucleotide sequence, as used herein, includes introducing the two exogenous nucleotide sequences into the host either simultaneously or sequentially. If introduction is sequential, it is preferable to introduce the methylase-encoding nucleotide sequence before introducing the unstable nucleotide sequence.

The present inventor has found that methylation of a nucleotide sequence can modulate the genetic stability of the sequence. In most cases examined, methylation of an unstable nucleotide sequence led to enhancement of the genetic stability of the nucleotide sequence, permitting the sequence to be replicated while maintaining sequence length. In a few cases, methylation of a nucleotide sequence led to reduced genetic stability of the nucleotide sequence.

The genetic stability of a nucleotide of interest (a selected nucleotide sequence) is modulated by replicating a nucleotide sequence comprising the selected nucleotide sequence in a host cell which methylates the selected nucleotide sequence or methylates sites in the flanking sequences, resulting in modulation of the genetic stability of the selected sequence. In a first embodiment, the method comprises co-transforming a host cell with the nucleotide sequence comprising the selected nucleotide sequence and with a nucleotide sequence encoding a methylase enzyme, and culturing the transformed host cell whereby the methylase enzyme is expressed and the selected nucleotide sequence, or a portion of the flanking nucleotide sequence, is methylated, thereby modulating the genetic stability of the selected nucleotide sequence.

The selected nucleotide sequence may be inserted into a recombinant vector such as a plasmid, cosmid, fosrnid, BAC or YAC. The selected nucleotide sequence may be an RNA sequence and the methylase enzyme employed is then an RNA methylase. In a preferred embodiment, the selected nucleotide sequence is a DNA sequence and the methylase enzyme employed is a DNA methylase.

In this embodiment, a host cell which lacks endogenous methylase activity is employed. Suitable host cells include bacterial cells, such as E, coli, yeasts and insect cells, such as cells from the fruit fly Drosophila melanogaster. Bacterial or other host cells should be strains which do not contain methylation restriction systems.

The methylase-encoding nucleotide sequence used to transform the host cell may be obtained from any source, either prokaryotic or eukaryotic. The DNA methylase employed may methylate CpG sites or other sites such as GpC or adenine.

Suitable DNA methylases include, but are not limited to, Alul, BamHI, Clal, dam, EcoRI, Haell, Hhal, Hpall, Hphl, Mspl, Pstl and Tagl and the mammalian methylases, DNMTI, DNMT3a, DNMT3b.

One example of a suitable methylase is the CpG DNA methylase from Spiroplasma sp. Strain MQ1 , designated herein Sss1 (Renbaum et al., (1990), Nucleic Acids Res., v. 18, p. 1145).

The methylase-encoding DNA may be introduced into the host cell either in an independently replicating recombinant vector or, preferably, by insertion into the genome of the host cell. Transformation may be carried out by any standard method, such as are described, for example, in Sambrook (latest edition).

Suitable recombinant vectors include plasmids, fosmids, cosmids, BAC's or YAC's. Suitable plasmids include, but are not limited to, pUC8, pUC19, pBR322, pBluescript and pSP64.

In a further embodiment, the method of modulating the genetic stability of a selected nucleotide sequence, such as a DNA sequence, comprises methylating a nucleotide sequence comprising the selected nucleotide sequence in vitro and introducing this methylated sequence into a host cell which has endogenous methylase activity, followed by culturing the host cell whereby the selected nucleotide sequence or flanking sites are methylated, thereby modulating the stability of the selected sequence.

Suitable host cells having endogenous DNA methylase include plant cells and mammalian cells. Mammalian cells may be primary cultures or established cell lines and may be obtained, for example, from hamsters, mice, rates, rabbits, pigs, cows and monkeys. Examples of suitable host cells include, but are not limited to, CHO cells and African green monkey kidney cells. The nucleotide sequence comprising the selected nucleotide sequence is methylated in vitro by any standard method, for example as described by the supplier of a commercially available DNA methylase.

The host cell is transformed with the methylated selected nucleotide sequence in the same manner as described above for a non-methylated sequence.

In a further embodiment, the invention provides a method for enhancing the genetic stability of an unstable nucleotide sequence by replicating a nucleotide sequence comprising the unstable sequence in a host cell which methylates the unstable sequence or methylates sites flanking the unstable sequence, resulting in enhancement of the genetic stability of the unstable sequence. As described above, the method may be carried out by co-transforming a host cell which lacks endogenous methylase activity with a nucleotide sequence comprising the unstable sequence and a methylase enzyme- encoding nucleotide sequence and culturing the host cell. In a further embodiment, also as described above, the method may be carried out by methylating the unstable sequence in vitro and then introducing the methylated sequence into a host cell having endogenous DNA methylase activity and culturing the host cell.

The method of the invention facilitates the cloning of unstable nucleotide sequences, including unstable DNA sequences, since stable replication of these sequences enables one to clone the sequences for further studies, for example for sequence determination.

Unstable nucleotide sequences may also be difficult to express recombinantly. The ability to enhance the genetic stability of unstable nucleotide sequences therefore also facilitates expression of such sequences and may assist in the production of expressed proteins from unstable sequences.

In addition, if a nucleotide sequence which has to be inserted into a cell as a means of gene therapy is an unstable sequence, the method of the invention facilitates the replication of the sequence to produce sufficient material for therapy, and permits enhancement of the genetic stability of the sequence after insertion into the cell to be treated.

It may also be desirable to create transgenic animals using as transgene a nucleotide sequence which has less than desirable genetic stability. The method of the invention permits one to stabilize the sequence by methylation prior to transforming the target animal.

The invention further provides a method for examining the effect of methylation on the genetic stability of a nucleotide sequence.

Plasmids containing various unstable elements were co-transformed into Eschenchia co/ with a plasmid, pAIT2, that expresses the Sssl CpG methylase which de novo methylates cytosine residues at the 5 position of CpG sites (Renbaum et al, 1990). While E. co// does not possess an endogenous CpG methylase, in this in vivo system virtually any plasmid DNA propagated within cells that also contain the pAIT2 plasmid will become methylated at most CpG sites. Hence, this system can be used to determine the effect of DNA methylation on the genetic instability of virtually any sequence during growth in living cells.

Using the method described herein, a DNA sequence propagated in a host cell transformed with a CpG DNA methylase-encoding nucleotide sequence becomes more than 95% methylated at every CpG site.

A bacterial strain with a methylase such as the Sssl methylase inserted into the bacterial chromosome permits the propagation of clones with various antibiotic resistances and various replication origins (obviating the problem of plasmid compatibility as well as co-isolation of the desired plasmid with the methlase-expressing plasmid). The cloning efficiency of certain GC-rich sequences, and those that are methylated, can be altered by the presence of the Sssl methylase vector, pAIT2 (i.e. when cloned alone or in a presence of in vivo CpG methylation).

The ability to stabilise unstable nucleotide sequences by the method of the invention also has clinical applications. For example, a pathogen such as HIV occurs in many variant forms and screening for variants is hampered by the general instability of the HIV genome. HIV variant screening may be facilitated by enhancing the genetic stability of the HIV nucleotide sequence by the method of the invention.

The invention also provides a method of mutagenising a DNA sequence comprising co-transforming a host cell with the nucleotide sequence and with a further nucleotide sequence encoding a DNA methylase enzyme and culturing the transformed host cell so that the methylase is expressed and brings about methylation of the DNA sequence, thereby altering its genetic sequence. The alteration may be, for example, a deletion, an insertion or a nucleotide substitution. Alternatively, the sequence may be used to transform a host cell having an endogenous methylase as described herein. The mutagenised sequence may be propagated and harvested by propagating the transformed host cell.

The invention further provides a method for promoting C → T mutatoins in a DNA sequence by methylating the sequence by the method of the invention; it is known that the methylation of cytosine residues favours such a C → T transition (Gonzalgo, 1997)

FRAXA

FRAXA is maternally inherited and is caused by the expansion of an unstable trinucleotide repeat (CGG)_n in the 5'-UTR of the FMR1 gene.

FRAXA (CGG)n instability is sensitive to the length and purity of the repeat and is likely to involve errors during DNA replication and/or DNA repair. Most of the non-affected human population have FRAXA alleles with 29-30 (CGG)_n repeats, which are stably transmitted. In normal individuals, the (CGG)_n repeat is interrupted by 1-3 AGG interruptions, which confer increased genetic stability to the repeat tract (Eichler et al., 1994), most likely by interfering with the formation of mutagenic intermediates, slipped stranded DNAs (Pearson et al., 1996; 1998). The stability threshold length at which increased genetic instability occurs is greater than or equal to 34 pure (CGG)_n repeats (Eichler et al., 1994). Tracts of 59-200 (CGG)_n repeats are highly unstable premutation alleles. FRAXA patients have 230-3000 (CGG)_n repeats. In FRAXA patients, the expanded (CGG)_n repeat and its neighboring CpG island are abnormally methylated (Oberle et al., 1991 ; Hansen et al., 1992).

Several lines of evidence suggest that the methylation status of the CpG island and of the (CGG)_n repeat are associated with somatic instability of the FRAXA repeats. Firstly, FRAXA individuals typically present distinct, large, methylated repeat expansions which are somatically stable throughout the patient's life. However, a subset of cases, the "high-functioning FRAXA patients", display both heterogeneous lengths of (CGG)_n expansions (ranging from full expansions down to normal lengths) and decreased amounts of abnormal methylation. in these individuals, usually the smallest mutations are those which are not methylated while the largest expansions are those that are methylated.

A second observation supporting a role for methylation in (CGG)_n instability is that all FRAXA males contain only premutation lengths of unmethylated (CGG)_n in the testes and sperm, while all other tissues contain full expansions of methylated (CGG)_n repeats (Reyniers et al., 1993; Malther et al., 1997). In both cases, the associated methylation status suggests that the somatic instability may be the result of the selective deletion of unmethylated (CGG)n repeats. There has been no direct evidence for a role of DNA methylation on (CGG)n repeat instability.

To test the effect of CpG methylation on the genetic stability of FRAXA (CGG)n repeats, a series of FRAXA patient-derived genomic clones were used in an E. coli based in vivo methylation system. The increased genetic instability of longer (CGG)_n repeats and their stabilization by AGG interruptions is recapitulated in both bacterial and yeast models (Shimizu et al., 1996; Hirst & White, 1998; White et al., 1999). The pFXA clones have repeat lengths above and below the stability threshold length of n=34 repeats (Table 1 ). This set of clones also includes differences in the purity of the repeat tract such that some clones differ only by the presence or absence of AGG interruptions. (The presence of an AGG interruption represents a lost methylatable CpG site within the (CGG)_n tract).

E.coli has been transformed with each of the pFXA plasmids (Table 1 ) either alone or with the pAIT2 plasmid. The Sssl expressing plasmid, pAIT2, contains a p15 replication origin and resistance to kanamycin, while each of the test plasmids used in this study contained ColE1 replication origins and resistance to ampicillin. This obviates plasmid incompatibility and permits selective growth of cells containing both or only one of the two plasmids. Transformed and co-transformed cells were propagated and sub-cultured as outlined in detail (Bowater et al., 1996). Following each sub-culturing, aliquots of cells were removed, plasmids harvested and the genetic stability of the pFXA clones determined by analyzing the length of the repeat-containing fragment by polyacryiamide gel electrophoresis (Fig. 1a). The stability of (CGG)_n tracts was very sensitive to the presence or absence of the methylase. In the absence of methylation, long pure (CGG)_n repeats (n=53) were very unstable, tending to delete (Fig. 1a and b). Strikingly, the co-transformation and in vivo methylation of the above- threshold stability length repeat resulted in increased stability, manifest as both a reduction in deletion events (Fig. 1c) and a reduction in the magnitude of the deletion (Fig. 1d). This increased stability was evident only when both the pAIT2 and pFXA clones were propagated together.

In several of the experiments, it was noted that, in cotransformed cells, early large deletion products were lost in subsequent sub-culturings, suggesting that in vivo methylation favoured the stable propagation of larger repeats.

The effect of CpG methylation on the stability of a (CGG)₅2 tract interrupted with two AGG units (pFXA9+9+32) was also investigated. In the absence of methylation, the long interrupted repeat displayed a few deletions, but was considerably more stable than the similar length pure tract pFXA53 (Fig. 1e). However, in the presence of methylation, the interrupted repeat tract of pFXA9+9+32 showed no deletions, indicating increased genetic stability. The direction of replication is known to affect the genetic stability of trinucleotide repeats during propagation in bacteria (Shimizu et al., 1996; Hirst & White, 1998) or yeast (White et al., 1999). The effect of methylation on the stability of a pFXA9+9+32(-), which has a repeat length and configuration identical to pFXA9+9+32 but is cloned in the opposite orientation, changing which strand of the repeat serves as the leading and lagging strand templates was also analysed. In the absence of methylation, pFXA9+9+32(-) was extremely unstable, resulting mostly in deletion products (Fig. 1e). This instability was greater than that observed for either pFXA53 or pFXA9+9+32. However, in the presence of methylation, the destabilizing effect of the direction of replication was reduced, and the pFXA9+9+32(-) repeat was stabilized consistently, showing both a reduction in deletion events and a reduction in the magnitude of the deletion. The effect of methylation on the stability of two short pure (CGG)_n repeats (n=20 and 21 ) replicated in the opposite direction, yielded similar results, stabilizing both clones and abolishing the destabilizing effect of replication orientation (Fig. 1e). Thus, in this system CpG methylation stabilized the FRAXA (CGG)_n repeats while, in the absence of methylation, the repeats showed a strong propensity to delete.

Myotonic Dystrophy

Myotonic dystrophy (DM) is associated with an unstable (CTG)_n repeat that is contained within a large CpG island. The unstable (CTG)n is in the 3'- UTR of the DMPK gene and is also in the promoter region of the SIX5 gene, immediately downstream of DMPK (Boucher et al., 1995). The instability of this repeat is very sensitive to repeat length and is likely to occur through aberrant DNA replication and/or DNA repair. Most of the non-affected human population have DM alleles with 15-25 (CTG)_n repeats, which are stably transmitted. Assymptomatic protomutation individuals have lengths ranging from 50-90 repeats which are genetically unstable. Typically, DM patients have 600-3000 (CGG)_n repeats and these lengths are highly unstable. While the unstable (CTG)_n does not contain the CpG methylase recognition site, recent evidence indicates that there is an association of abnormal hypermethylation of the surrounding CpG island with both large (greater than 1000 repeats) (CTG)_n expansions as well as earlier age of disease onset and severity of symptoms (Steinbach et al., 1998). The role, if any, of DNA methylation in DM (CTG)_n instability was unknown prior to the work of the present inventor. To test the effect of CpG methylation on the genetic stability of DM

(CTG)_n repeats a series of DM clones were used in the in vivo methylation system of the present invention and evaluated (Table 1 ). The DM clones had repeat lengths above and below the genetically unstable protomutation length of 50-90 repeats, up to disease-associated lengths of 100 repeats (Table 1 ). Cotransformation of Sssl expressing plasmid pAIT2 with a myotonic dystrophy clone containing (CTG)₅₀ resulted in an increase in instability, manifest as both repeat deletions and expansions (Fig. 2a to c). Furthermore, when propagated in the presence of in vivo methylation, the DM plasmids tended to delete fewer repeats (Table 1 ).

In some co-transformations with (CTG)₃_, (CTG)₅o, (CTG)₈₃, and (CTG)ioo, expansion products were observed, detectable by ethidium bromide staining (Fig. 2B and 2C). Individual products of expansion events were isolated and length changes were confirmed by sequencing to be increases of integral numbers of repeats (increases of +13 to +69, +18 to +28, +98 to +116, and +87, respectively) (Table 1 , and data not shown). These expansion events were only ever observed with DM plasmids propagated in the presence of pAIT2. This suggests that, in the presence of the Sssl methylase and/or possibly the in vivo methylation of flanking CpG sites, the (CTG)_n repeat shows a pronounced ability to expand. Similar to the FRAXA (CGG)n results, in several experiments (Fig. 2) it was noted that, in cotransformed cells, early large deletion products were lost in subsequent sub-culturings, suggesting that in vivo methylation favoured the stable propagation of larger (CTG)_n repeats. The genetic stability of plasmid-borne (CTG)n'(CAG)_n repeats is sensitive to the orientation of the repeat relative to the replication origin (Kang et al., 1995; Freudenreich et al., 1998). The effect of methylation on the stability of two (CTG)-ιoo tracts cloned in the stable [(CTG)-ιoo] and unstable [(CTG)ioo(-)] orientations was analyzed. In the absence of methylation, the (CTG)ι_o(-) was extremely unstable, resulting in large deletions leaving only a few repeats (Fig. 2d). However, in the presence of pAIT2, the effect of orientation appears to be absent, with the stabilization of the (CTG)₁₀o(-)- In vivo methylation stabilized the (CTG)_n repeats by reducing large deletions and permitting expansion events.

Moreover, the DM results indicate that the methylation status of adjacent sequences can affect the genetic stability of a contiguous sequence. Using the same parameters used to identify mammalian CpG islands (Boucher et al., 1995), sequence analysis of the cloning vectors (including pBR322, pBluescript, pUC8, pUC19, and pSP64) revealed that each contain multiple CpG islands (data not shown). The methylation status of these vector CpG islands may alter the genetic stability of the cloned unstable elements. Notably, in the human context, many of the unstable elements investigated, including the DM locus, are contained within or proximal to CpG islands (Brock et al., 1999). The methylation status of these CpG islands may alter the genetic stability of adjacent repeats.

α3'HVR

The analysis of the present system was extended to the 'hypervariable region', α3'HVR, which is within a GC-rich isochore 8-kb downstream of the human α1-globin gene (Jarman et al., 1986). This minisatellite has a 17 base repeat unit that contains two CpG sites (Table 1 ) and displays an unusually high degree of length variability (90-100% heterozygosity), with ranges in the population from 15-450 repeats. The p3' HVR.64 clone contains 228 repeats and is extremely unstable, tending to delete during propagation in bacteria (Jarman et al., 1986). Propagation of this plasmid in the presence or absence of Sssl had a striking effect on the stability of this repeat (Fig. 4A). Restriction digests of the starting material containing all 228 repeats resulted in a 4-kb band. Following sub-culturing, this had deleted to several discrete lengths (Fig. 4A). In the presence of Sssl methylase, this repeat was consistently more unstable, rapidly and completely deleting down to only 20 repeat units (Fig. 4A). In vivo methylation enhanced the instability (deletions) of the α3'HVR repeats.

JCF

At the molecular level, ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) patients display juxtacentromeric chromosomal abnormalities and abnormal hypomethylation of various classical repeat sequences (satellites II and III, alpha satellites and Alu repeats). ICF patients have mutations within the gene encoding the ofe novo methylase DNMT3a. The human DYZ1 repeat, a satellite III sequence, is composed of 800-5000 direct copies of a ~3.4-kb EcoRI fragment, which itself is composed of a tandemly repeated pentanucleotide (Nakahori et al., 1986). The pHY10 clones (R and L designating both cloning orientations) contain a complete single 3564 bp EcoRI unit, consisting of 713 tandem repeats of TTCCA and its single nucleotide derivatives including the CpG-containing TTCGA (Table 1 ). This repeat is normally methylated in differentiated tissues and unmethylated in tissues from ICF patients (Jeanpierre et al., 1993). It remains to be determined whether any of the repeats displaying altered methylation in ICF also display repeat length alterations. The effect of methylation was tested on the stability of both pHY10R and pHY10L in the in vivo methylation system. Neither of the DYZ1 clones displayed any instability alone or in the presence of CpG methylation (Fig. 4B).

HNPCC

Many cancers, particularly those of Hereditary Non-Polyposis colorectal cancer (HNPCC), are associated with altered regulation of DNA methylation dynamics (Ahuja et al., 1997; Toyota et al., 1999). A characteristic of HNPCC tumors is genome-wide instability at microsatellites (Thibideau et al., 1993, Aaltonen et al., 1993, lonov et al., 1993) and is most commonly diagnosed at dinucleotide repeats (Deitmaier et al., 1997). The effect of CpG methylation on the genetic stability of plasmids containing (TC)₃₇, or (CA)₃₀- or (GC)ι₃, (the latter containing the recognition site CpG) was tested. These dinucleotide repeats display length instability both in humans, yeast, and in bacteria (Freund et al., 1989; Strand et al., 1993).

In the absence of methylation, the stability of both (TC)37 and (CA)30 was sensitive to their orientation relative to the replication origin (Fig. 3a and 3b) and over multiple sub-culturings tended to rapidly delete in the (-) orientation. Similariy the (GC)13 plasmid was also unstable, tending to delete (Fig. 3c). In the presence of in vivo methylation, each of the dinucleotides (TC)37, (TC)37(-), (CA)30, (CA)30(-) and (GC)13 were completely stabilized and displayed no deletion products, effectively eliminating the deleterious effect of replication orientation. Moreover, in the presence of methylation, the (GC)13 plasmid displayed products of expansion events (Fig. 3c). Thus, methylation also affected the stability of dinucleotide repeats, which supports the suggestion that cancer-associated alterations of methylation may exacerbate mutation rates. Furthermore, the DM (CTG)n results and the (TC)n and (CA)n results together demonstrate that in vivo methylation can modify the genetic stability of sequences lacking methylatable sites, indicating that methylation of adjacent sequences can alter the stability of contiguous sequences.

Mammalian Cells

To determine the effect of CpG methylation upon repeat stability in living mammalian cells, the SV40 viral replication system was used. Placement of the SV40 virus replication origin (SV40-or/) into a repeat- containing clone allows it to replicate either within primate (COS1 ) cells expressing T-antigen (Gluzman, 1980) or in the presence of primate cell extracts and T-antigen (Tag) (Stillman, 1980; Roberts et al., 1988). The SV40-or/ was inserted into the pFXA53 clone (Fig. 5). In this clone, the CGG repeat is in the more stable orientation with respect to bacterial replication, while mammalian SV40-directed replication would predictably be in the more unstable orientation.

Transfection of in vitro methylated (Sssl) and un-methylated DNAs into COS1 cells allowed for replication of chromatin-assembled templates (Cereghini & Yaniv, 1984). Following transfection (48 hours) replication products were isolated (Hirt, 1967) and digested with Dpnl. Material resistant to Dpnl cleavage represents products of complete replication in mammalian cells. Mutation analysis of the mammalian replicated DNAs was performed by transformation of Dpnl-resistant material into bacterial cells and the repeat length of DNA prepared from single colonies was compared to that of starting length plasmid by polyacryiamide gel electrophoresis. Following replication in COS1 cells, the methylated pFXA53-SVB was more stable, experiencing fewer deletions events relative to the un-methylated form (Table 2). It was confirmed that changes in electrophoretic migration were due to changes in numbers of repeats by southern blot hybridization specific for (CGG)n repeats and through DNA sequencing of individual deletion products (data not shown). CpG methylation stabilized the FRAXA (CGG)n repeats in this primate cell system.

To investigate whether the difference in genetic stability between the methylated and un-methylated templates was due to chromatin packaging, the DNAs were replicated in vitro in the presence of cytoplasmic extracts and TAg, where chromatin does not form (Stillman, 1980). Similar to the transfection results, the methylated template was more stable compared to its un-methylated form (data not shown). Thus, it seems unlikely that the effect of methylation upon stability is mediated by chromatin packaging.

It has been demonstrated that the genetic stability of various DNA sequences can be modified by the CpG methylation status of or around the loci, suggesting a direct role of methylation in mutagenesis. The altered instability of test plasmids was only observed when they were co-transformed with the Sssl expressing pAIT2 vector. Co-transformation of test plasmids with the vector minus the Sssl gene (pACM184) did not alter the genetic instability of any plasmids (data not shown). Thus, the modification of genetic instabilities is most likely due to the in vivo expression of the Sssl CpG methylase and/or due to the in vivo methylation of CpG sites. In vitro methylation of plasmids prior to transformation die not alter their genetic stability propagated in vivo (data not shown), indicating that the effects of methylation are transmitted by ongoing in vivo methylation.

The effect of methylation was specific to the cloned unstable elements, as co-propagation of the base vectors pUC8, pUC19, pBR322, pBluescript, or pSP64, along with pAIT2 did not result in any alterations, insertions or deletions in their sequences (data not shown). Furthermore, as an internal control to each experiment the pAIT2 vector was never observed in any instance to display sequence alterations, indicating that the effect of CpG methylation was specific to the cloned unstable elements.

The altered genetic stability of unstable elements that do not contain CpG sites suggests that either 1 ) methylation of flanking sequences may induce alterations in the genetic stability of downstream or upstream DNAs; or 2) the altered stability may be due to some trans-acting factor, possibly the Sssl methylase itself (Yang et al., 1995). These two possibilities are not mutually exclusive. Notably, many of the unstable elements investigated are contained within or proximal to CpG islands, and the epigenetic status of these islands may alter the genetic stability of proximal regions (Brock et al., 1999). Many of the vectors containing the unstable elements (including pBR322, pBluescript, pUC8, pUC19, and pSP64) are rich in CpG sites, and when analyzed by usual parameters (Boucher et al., 1995) each contain multiple CpG islands (data not shown). The in vivo methylation system described herein permits the cloning of

"unclonable" sequences or the stabilizing of unstable sequences. Preliminary results reveal that long GC-rich cosmid clones (~40 kb long) are genetically more stable when propagated with the Sssl methylase expressing pAIT2 (data not shown).

Hot Spots

Expansions of (CTG)_n and (CGG)_n trinucleotide repeats have been demonstrated in yeasts deficient in rad27, the homologue of FEN-1 enzyme (Freudenreich et al., 1997; White et al., 1999), which participates in Okazaki processing, DNA repair and DNA recombination (Lieber, 1997). Expansions of (CTG)_n have been observed in bacteria deficient in the SbcCD endonuclease (Sarkar et al., 1999). Both the rad27 and SbcCD mutants have genome-wide effects on genetic stability, and neither of these are known to be deficient in any patients suffering from either DM or FRAXA. Exactly what determines the location of a mutation 'hot spot' is not known. For example, what signals the genetic instability of the FRAXA (CGG)_n repeat in one individual while, in the same individual, the FRAXE or the FRAXF (CGG)_n repeats are genetically stable. Were the instability mediated by a trans-acting (replication or repair) factor, one would expect genome-wide instability, as is the case for mismatch repair defects found in Hereditary Non Polyposis Colorectal Cancer tumors. Parenthetically, it is to be noted that not all dinucleotides of the same sequence display the same instability in HNPCC tumors (Deitmaier et al., 1997). The present results suggest that the epigenetic effect of altered CpG methylation may act as such a signal.

FRAXA Biology Remarkably, for FRAXA both the expansion of the premutation to the full mutation and the transmission of the disease occurs only when maternally transmitted. Evidence indicates that the initial expansion event of the FRAXA (CGG)n repeat occurs meiotically during maternal oogenesis (Malther et al., 1997). This only partially explains the parent of origin effect. The means through which premutation or full mutation FRAXA males have only premutation lengths in their sperm is not clear. Some unknown mechanism is selectively allowing the male germ cells to delete the expanded FRAXA (CGG)n repeats (Reyniers et al., 1993; Malther et al., 1997). The present results suggest that altered abnormal methylation may mediate this process. The present results also shed light on the association of methylation mosaicism with the increased somatic instability of the (CGG)_n repeat in "high- functioning" FRAXA patients: That is, following the initial expansion event, the expanded alleles that escaped subsequent abnormal hypermethylation may have undergone continued somatic instability (manifest as deletions), while for methylated expanded alleles the instability process was halted. The results suggest that this differential somatic instability may be mediated by differential abnormal methylation of the expanded repeats.

Mammalian systems Bacterial and mammalian systems have been used to directly investigate the effect of methylation on the genetic stability of mammalian sequences. The comparable results obtained in bacterial and primate cells may indicate a general effect of CpG methylation on stability and sheds light on a possible mechanism through which methylation may modify repeat stability. The similar effect of methylation on genetic stability in such evolutionarily divergent cells, which have different proteins for methylation, replication, repair and chromatin packaging, argues against a specific protein requirement for the effect. Specifically the effect on stability can be transmitted by methylation by enzymes other than Sssl, since while mammalian cells do contain methylases Xu et al., 1999) they do not contain Sssl. The effect of methylation may be transmitted through a factor that is common to both systems: the methylation status of the DNA (possibly its increased melting temperature) which may equally hinder replication fork progression in bacterial and mammalian cells. The biophysical attributes of the methylated DNAs specifically their increased melting temperature (Collins & Meyers, 1987) may affect both the rate and fidelity of DNA polymerase synthesis and of 3'→5' exonuclease proofreading. The methylation status may favor or disfavor the ability to form mutagenic intermediates such as slipped structures during replication — in this way methylation may enhance or diminish mutagenesis. Notably, methylated DNAs have increased melting temperatures (Collins & Meyers, 1987) and methylation can enhance or impair the ability of certain sequences to form non-B-DNA structures such as melted DNA, bent DNA, cruciforms, Z-DNA, triplex-DNA, and higher-order DNA-DNA interactions (Zacharias, 1993). Importantly the effects of methylation on DNA structure are both sequence- and context-dependent and its effects can extend beyond the methylated region; the latter may explain the modified genetic stability of un-methylatable sequences flanked by methylated DNAs. Methylation may modify genetic stability through alterations in repairability — depending upon methylation status slipped repeats may escape repair or be repaired in an error-prone fashion, either of which could result in repeat length changes. The results with multiple repeat sequences clearly demonstrate that CpG methylation can modify genetic stability in living cells, thereby establishing the existence of novel methylation-dependent mechanisms of mutagenesis.

The epigenetic effect of altered CpG methylation may act as a signal for a mutation 'hot spot'. Methylation status may contribute to the locus- specific instability that is common to many diseases such as DM and FRAXA. Rather than the result of frans-acting factors such as Fen1 (Freudenreich et al., 1998; Lieber 1997) and SbcCD (Sarkar et al., 1998) which have genome- wide effects on genetic stability (Lieber, 1997; Leach, 1994), one might expect that c/s-acting factors such as sequence or epigenetic alterations would predispose to site-specific instability, such as that exhibited in DM or FRAXA patients. The results reveal that in addition to DNA sequence and replication direction, altered locus methylation status can act as a signal for a mutation hot spot and modify genetic stability. Such epigenetic modification of mutation disposition has broad implications for genome stability.

Examples The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

Methods of chemistry, biochemistry and molecular biology referred to but not explicitly described in this disclosure and examples are reported in the scientific literature and are well known to those skilled in the art.

Materials

Bill Jack at New England Biolabs provided pAIT2 and pACM184.

Dr. Y. Nakahori provided pHYIOR and pHYIOI (Nakahori et al., 1986). pα3'HVR was provided by L.W. Coggins, originally from A.R. Higgs (Jarman et al., 1986). pET, containing (CA)30 and p(GA)37 were provided by Dr. B. Johnston (Stanford, CA), these were originally from Dr. F. Strauss (Science) and Dr. Efstratiadis (ref.), respectively. p(GC)6 and p(GC)13 were provided by Drs. RP Fuchs and M. Bichara (Freund et al., 1989).

Methods Plasmids harbouring unstable elements were transformed or co- transformed with the Sssl CpG methylase expressing plasmid pAIT2 into competent E. coli, and propagated as described (Bowater et al., 1997). Briefly, transformations were performed with an excess number of cells to DNA molecules, thus favoring transformation of a single cell by an individual plasmid, or plasmids in the case of co-transformations. Cultures were rigorously maintained in log phase [monitored by optical density (O.D.₆₀0 = 0.7)]. Subculturing was done by innoculating fresh medium with a 10⁷ dilution of the previous culture at an of O.D.₆oo = 0.3-0.8. At each dilution, the cells from a portion of the culture were harvested, and plasmids purified by Magic Mini Preparation (Promega), with the inclusion of a proteinase K digestion. DNAs were restriction digested and analysed by polyacryiamide or agarose gel electrophoresis. No significant difference was found in results between plasmids that had or had not been gel-purified prior to transformation.

Each of the test plasmids (Table 1 ) were transformed alone or with the Sssl expressing plasmid, pAIT2. pAIT2 contains a p15 replication origin and resistance to kanamycin, while each of the test plasmids used in this study contained ColE1 replication origins and resistance to ampicillin. This obviates plasmid incompatibility and permits selective growth of cells containing both or only one of the two plasmids. Following various times of bacterial propagation, as outlined in detail (Bowater et al., 1996), the genetic stability of test clones were determined by analyzing the length of the repeat-containing fragment by polyacryiamide gel electrophoresis (Fig. 1 ). The CpG methylation status of all test plasmids that were co-transformed with pAIT2 were monitored by methyl-sensitive (Ac/I and Fnu4HI) restriction digestion (data not shown). To avoid degradation plasmids were transformed into cells deficient in the methyl-specific restriction systems mcrA, mcrCB, mrr. All experiments presented were performed with both ER1821 and XL1-Blue MR, and some experiments were done in SURE, and Stbl2 cells. No drastic difference between cell lines was observed.

This in vivo methylation system can be used with other genetic strains of E. coli that are deficient in other genes known to contribute to genetic stability (for instance the mismatch repair genes, mutS, mutH, and/or mutL). Such mutants may prove to increase or decrease the effect of in vivo CpG methylation on genetic stability.

For analysis of mammalian stability, isolated Dpnl-resistant replication products were transformed into E. coli, DNAs were prepared from individual bacterial colonies — each derived from an individual product of mammalian replication. DNAs were analysed for repeat changes by gel electrophoresis, and products analysed by gel electrophoresis and repeat-specific hybridisation. This mutation analysis permitted the identification of individual products of repeat length changes and their characterization (sequence and magnitude of the change). Analysis of many (40-150) single colonies from multiple experiments yielded a relative frequency of mutations for a given template (+/- methylation). Precautions were taken to minimize instability introduced by the bacteria during re-transformation: 1 ) in the SV40 construct the trinucleotide repeats were cloned in the stable bacterial orientation; 2) the number of bacterial cell generations required for mini-plasmid preparation is 4-6 — far fewer than the 25-150 required to observe bacterial induced repeat instability (see Fig. 1 ). Under these conditions the background instability contributed by bacteria for each template was minimal (ranging from 4-7%). Background was subtracted from the mammalian mutation frequencies essentially as described by Kunkel and colleagues (Roberts et al., 1988). These measures permitted an accurate determination of the contribution of mammalian-mediated instability.

Example 1 : Effect of methylation on the genetic stability of FRAXA (CGG)n repeats. The pFXA53 plasmid containing (CGG)53 repeats (Table 1) was propagated in E. coli in the absence or presence of the Sssl CpG methylase expressing plasmid pAIT2. Following each of three sub-culturings (each representing 25 generations), an aliquot of cells was harvested, plasmids isolated and restriction digested and analyzed by 4% polyacryiamide gel electrophoresis and ethidium bromide staining. As the number of generations giving rise to plasmid DNA was equivalent in the presence or the absence of in vivo methylation, a direct comparison of stability was possible. To test for repeat length changes, DNAs were digested with Kpnl and Sacl which liberates the (CGG)n repeat-containing fragments from the pFXA clones, each of which are resolvable from pAIT2 and vector bands. The starting lengths of (CGG)n are indicated in Figure 1a by dark arrowheads; all other faster migrating DNAs are products of (CGG)n deletion events indicated by brackets. The pAIT2 and the FRAXA vector are indicated by light arrowheads. The gel of Figure 1a is representative of 5 or more independent experiments.

To confirm that products were due to changes in repeat numbers, the gel shown in Figure 1 , panel a, was electrotransferred to nylon membrane and hybridized to radiolabeled (CGG)10 oligonucleotides and exposed for autoradiography, as shown in Figure 1 b. Individual products were also analyzed by DNA sequencing.

The effect of methylation (-/+ pAIT2) on repeat length stability was determined by densitometric analysis of multiple experiments as described in Kang et al. ((1995). The results are shown in Figure 1c. The open area represents the percentage of material with the starting length of repeats while the shaded area represents the percentage of deletion products.

The magnitude of repeat loss was determined by streaking on plates cells from each sub-culturing of a test plasmid in the absence or presence of methylation (-/+ pAIT2), isolation of >20 single colonies and analysis of their DNA. The analysis of pFXA53 is shown in Figure 1d. Deletion sizes for the first, second and third sub-culturings are shown by open, shaded, and cross- hatched bars, respectively.

FRAXA plasmids containing the indicated lengths, purities and orientations of (CGG)n tracts (Table 1 ) were propagated through three sub- culturings in E. coli in the absence or presence of methylation (-/+ pAIT2). Following each sub-culturing cells were harvested and (CGG)n repeat length changes analyzed as above. The trend through the three sub-culturings is reflected by the final sub-culture; hence only the third sub-culturing for each plasmid is shown in Figure 1e. This gel is representative of 5 or more independent experiments. Products are labeled as in Figure 1 , panel a.

Example 2: Effect of methylation on the genetic stability of DM (CTG)n repeats.

The DM plasmid containing (CTG)83 repeats (Table 1 ) was propagated in E. coli in the absence or presence of the Sssl CpG methylase expressing plasmid pAIT2. Following three sub-culturings (each representing 25 generations) an aliquot of cells was harvested, plasmids isolated and restriction digested and analyzed by 4% polyacryiamide gel electrophoresis and ethidium bromide staining. To test for repeat length changes DNAs were digested with Sacl and Pst\ which liberates the (CTG)n repeat-containing fragments from the DM clones. These fragments are resolvable from pAIT2 and vector bands. Results are shown in Figure 2a. The starting lengths of (CTG)n are indicated by dark arrowheads, slower migrating (CTG)n expansion products are indicated by open arrowheads, and faster migrating DNAs are products of (CTG)n deletion events indicated by brackets. The pAIT2 and the DM vector are indicated by light arrowheads. This gel is representative of 5 or more independent experiments.

To confirm that products were due to changes in repeat numbers, the gel shown in Figure 2a was electrotransferred to nylon membrane and hybridized to radiolabeled (CTG)15 oligonucleotides and exposed for autoradiography, as shown in Figure 2b. Individual products were also analyzed by DNA sequencing.

DM plasmids containing the indicated (CTG)n tracts (Table 1 ) were propagated through three sub-culturings in E. coli in the absence or presence of methylation (-/+ pAIT2). Following each sub-culturing cells were harvested and (CTG)n repeat length changes analyzed as above. The effect of methylation through the three sub-culturings of DM plasmids was similar to that observed for (CTG)83 (Figures 2 a and b). This trend is reflected in the final sub-culture, hence only the third sub-culturing in the presence of methylation is shown for each plasmid in Figure 2c. Expansion products, indicated by hollow arrowheads were observed only in the presence of methylation. For each experiment repeat length changes of individual expansion and deletion products were confirmed by DNA sequencing.

Plasmids containing 100 CTG repeats cloned in the stable [(CTG)100] and unstable [(CTG)100(-)] orientations (Table 1) were sub-cultured three times in the absence or presence of methylation (-/+ pAIT2), and (CTG)n repeat length changes analyzed as above. While the deleterious effect of replication orientation on this long tract is not evident in the third subculturing, the stabilizing effect of methylation is evident; hence only the third sub- culturing is shown in Figure 2d. Products are labeled as in Figure 2a. These gels are representative of 5 or more independent experiments.

Example 3: Effect of methylation on the genetic stability of dinucleotide repeats.

Plasmids containing the indicated dinucleotides (TC)37, (CA)30, and (GC)13 and their cloning orientations (Table 1 ) were propagated in E. coli for three sub-culturings (each representing 25 generations) in the absence or presence of the Sssl CpG methylase (-/+ pAIT2). Cells were harvested, plasmids isolated and restriction digested and analyzed by polyacryiamide gel electrophoresis (PAGE) and ethidium bromide staining. The trend through the three sub-culturings is reflected by the final sub-culture; hence only the third sub-culturing is shown for each plasmid in Figure 3. The starting length of each repeat is indicated by dark arrowheads, slower migrating repeat expansion products are indicated by open arrowheads, all other faster migrating DNAs are products of deletion events. (GC)n expansions are indicated by open arrowheads. For each experiment, repeat length changes of individual expansion and deletion products were confirmed by DNA sequencing. Figure 3a, (TC)37 and (TC)37(-) digested with Bam \/Nde\ analyzed on 8% PAGE. Figure 3b, (CA)30 and (CA)30(-) digested with SamHI/H/ndlll analyzed on 8% PAGE. Due to the opposite cloning orientation and the polylinker the (CA)30 restriction fragments migrate faster than their sister (CA)30(-) restriction fragments. Figure 3c, (GC)13 digested with SamHI/H/ndlll analyzed on 20% PAGE. Gels in panels a, b, and c are representative of 5 or more independent experiments.

Example 4: Effect of methylation on the genetic stability of minisatellite and satellite 3 repeats.

Plasmids pα3'HVR and pHYIOL containing the minisatellite 3' of the alpha globin gene and the DYZ1 satellite 3 repeat, respectively (Table 1 ) were propagated for three sub-culturings in E. coli in the absence or presence of the Sssl CpG methylase expressing plasmid pAIT2. Cells were harvested, plasmids isolated and restriction digested and analyzed by 0.7% agarose gel electrophoresis and ethidium bromide staining. The trend through the three sub-culturings is reflected by the final sub-culture; hence only the third sub- culturing is shown for each plasmid in Figure 4.

pα3'HVR digested with BamHVHind Ill/Seal is suitable to test for repeat length changes as it liberates the minisatellite-containing fragments; each of which are resolvable from vector and pAIT2 and vector restriction fragments, as shown in Figure 4a (indicated by light arrowheads). The starting length of the 228 repeats is indicated by a dark arrowhead; all other faster migrating DNAs are products of deletion events, indicated by arrows.

Plasmid pHYIOL digested with Sspl/H/ndlll is suitable to test for repeat length changes as it liberates the satellite-containing fragment — which is resolvable from pAIT2 and restriction fragments as shown in Figure 4b (indicated by light arrowheads). The starting length of the 713 repeats is indicated by a dark arrowhead. The sister clone pHYIOR, which is identical but cloned in the opposite orientation, yielded identical results (not shown). Gels in panels a and b are representative of 5 or more independent experiments.

Example 5: Effect of methylation on the genetic stability of FRAXA (CGG)n repeats in a primate system.

The Spnl-Hndlll fragment of the SV40 virus, which contains the replication origin, was cloned into the Bgl\ site of the pFXA53 plasmid containing (CGG)53 repeats (Figure 5a and Table 1).

Following either transfection into COS1 cells, or in vitro replication, the mammalian replication products were analyzed for repeat length alterations: isolated Dpnl-resistant replication products were transformed into E. coli, DNAs were prepared from individual bacterial colonies - each derived from an individual product of mammalian replication. DNAs were analysed for repeat changes by gel electrophoresis, and repeat-specific hybridization (not shown).

Following the analysis of multiple colonies, frequencies of deletion events were scored and. Chi-square analysis revealed a significant difference between the stability of methylated and un-methylated templates either in living cells (p=0.014) or in vitro (p=0.00088). The results are shown in Table 2. Although preferred embodiments have been described herein in detail it is understood by those skilled in the art that variations can be made hereto without departing from the scope of the invention.

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Table ! Sequences/Clones Analyzed

Clone repeat repeat replication effect of name length configuration orientation methylation

Fragile X Mental Retardation pFXA53 (CGG)53 pure + E

PFXA9+9+32 (CGG)52 9+9+32 + E pFXA9+9+32(-) (CGG)52 9+9+32 - E pFXA20 (CGG)20 pure + E pFXA21 (-) (CGG)21 pure - E

Myotonic Dystrophy

PRW3211 (CTG)17 pure + E

PRW2180 (CTG)30 pure + E, exp

PRW3213 (CTG)50 pure + E, exp

PRW3214 (CTG)83 pure + E, exp

PRW3245 (CTG)100 pure + E, exp

PRW3246 (CTG)100(-) pure - E, exp

Cancer-Associated Instability p(TC)37 (TC)37 pure + E

P(TC)37(-) (TC)37 pure - E p(CA)30 (CA)30 pure + E

P(CA)30(-) (CA)30 pure - E pUC(GC)13 (GC)13 pure NA E, exp

Minisatellite p°c3'HVR 64 (AACAGCGACACGGGGGG)228 homogenous R

Immunological Centromeric Facial Abnormalities

PHY10L/DYZ1 (TTC A)713 homogenous N pHY10R/DYZ1 (TTC^C/_GA)713 homogenous N

Characteristics of unstable sequences The plasmid name, sequence of the repeat unit and the total number of repeats are indicated The purity of the repeat configuration is also indicated For the FRAXA clones the + indicates an AGG interruption¹² The α3'HVR repeat is highly homogenous with some repeat units differing at the 3' end²⁵ Only the major repeat unit variants are shown for the DYZ1 repeat²⁵ The cloning orientation of the insert relative to the unidirectional plasmid replication origin is indicated by + and -, reflecting the stable and the unstable orientation respectively in bacteria The repeat sequence shown represents the template strand for leading strand synthesis at the replication fork in the stable orientation (+) The 3'HVR could only be cloned in one orientation²⁵ Since the sequence in both strands of the (GC)n repeat are identical, cloning orientation and hence replication direction was not relevant — NA refers to not applicable For more details of the clones see Supplementary Information The effect of in vivo methylation on sequence stability are summarized in the rightmost column, where E, R and N refer to ennanced stability reduced stability and no effect relative to the absence of methylation, respectively E, R and N were determined from >5 experiments for each sequence by product analysis following multiple conrolied sub-culturings (for a representative example see Fig 1 a-d) Enhanced stability re.erε to tewer deletion events and smaller sizes o^* deletion, while reduced stability refers to mcreaseα αeietion events and larger αeietion sizes The 'exp ' indicates that methylation-dependent expansions were observed (see text) Table 2

colonies methylation frequency (%)

PFXA53-SVB analysed status deletions

103 42.72

COS1 transfection

79 48.10

43 37.21 in vitro replication

42 47.62

Claims

We claim:

1. A method of modulating the genetic stability of a selected nucleotide sequence, the method comprising the steps of:

(a) providing a nucleotide sequence comprising the selected nucleotide sequence;

(b) replicating the nucleotide sequence in a host cell capable of methylating the selected nucleotide sequence or a portion of the nucleotide sequence flanking the selected nucleotide sequence, thereby modulating the genetic stability of the selected nucleotide sequence.

2. The method of claim 1 wherein step (b) comprises:

(i) co-transforming a host cell with the nucleotide sequence comprising the selected nucleotide sequence and a nucleotide sequence encoding a methylase enzyme; and

(ii) culturing the transformed host cell whereby the methylase enzyme is expressed and the selected nucleotide sequence, or a portion of the nucleotide sequence flanking the selected nucleotide sequence, is methylated, thereby modulating the genetic stability of the selected nucleotide sequence.

3. The method of claim 1 where step (b) comprises:

(i) methylating the nucleotide sequence comprising the selected nucleotide sequence;

(ii) providing a host cell comprising an endogenous methylase enzyme;

(iii) transforming the host cell with the methylated nucleotide sequence of step (i); and (iv) culturing the transformed host cell whereby the selected nucleotide sequence, or a portion of the nucleotide sequence flanking the selected nucleotide sequence, is methylated, thereby modulating the genetic stability of the selected nucleotide sequence.

4. A method of enhancing the genetic stability of a selected unstable nucleotide sequence, the method comprising the steps of:

(a) providing a nucleotide sequence comprising the selected unstable nucleotide sequence;

(b) replicating the nucleotide sequence in a host cell capable of methylating the selected unstable nucleotide sequence or a portion of the nucleotide sequence flanking the selected unstable nucleotide sequence, thereby enhancing the genetic stability of the selected unstable nucleotide sequence.

5. The method of claim 4 wherein step (b) comprises:

(i) co-transforming a host cell with the nucleotide sequence comprising the selected unstable nucleotide sequence and a nucleotide sequence encoding a methylase enzyme; and

(ii) culturing the transformed host cell whereby the methylase enzyme is expressed and the selected unstable nucleotide sequence, or a portion of the nucleotide sequence flanking the selected unstable nucleotide sequence, is methylated, thereby enhancing the genetic stability of the selected unstable nucleotide sequence.

6. The method of claim 4 wherein step (b) comprises: (i) methylating the nucleotide sequence comprising the selected unstable nucleotide sequence;

(ii) providing a host cell comprising an endogenous methylase enzyme;

(iii) transforming the host cell with the methylated nucleotide sequence of step (i); and

(iv) culturing the transformed host cell whereby the selected unstable nucleotide sequence, or a portion of the nucleotide sequence flanking the selected unstable nucleotide sequence, is methylated, thereby enhancing the genetic stability of the selected unstable nucleotide sequence.

7. A method for mutagenising a nucleotide sequence, the method comprising the steps of:

(a) providing a nucleotide sequence;

(b) replicating the nucleotide sequence in a host cell capable of methylating the nucleotide sequence, thereby mutagenising the nucleotide sequence.

8. The method of claim 7 wherein step (b) comprises:

(i) co-transforming a host cell with the nucleotide sequence and a nucleotide sequence encoding a methylase enzyme; and

(ii) culturing the transformed host cell whereby the methylase enzyme is expressed and the nucleotide sequence is methylated, thereby mutagenising the nucleotide sequence.

9. The method of claim 7 wherein step (b) comprises: (i) methylating the nucleotide sequence; (ii) providing a host cell comprising an endogenous methylase enzyme;

(iii) transforming the host cell with the methylated nucleotide sequence of step (i); and

(iv) culturing the transformed host cell whereby the nucleotide sequence is methylated, thereby mutagenising the nucleotide sequence.

10. A method for methylating a nucleotide sequence, the method comprising replicating the nucleotide sequence in a host cell capable of methylating the nucleotide sequence.

11. The method of claim 10 comprising the steps of:

(i) co-transforming a host cell with the nucleotide sequence and a nucleotide sequence encoding a methylase enzyme; and

(ii) culturing the transformed host cell whereby the methylase enzyme is expressed and the nucleotide sequence is methylated.

12. The method of claim 10 comprising the steps of:

(i) methylating the nucleotide sequence;

(ii) providing a host cell comprising an endogenous methylase enzyme;

(iii) transforming the host cell with the methylated nucleotide sequence of step (i); and

(iv) culturing the transformed host cell whereby the nucleotide sequence is methylated.

13. The method of any one of claims 1 to 6 wherein the selected nucleotide sequence is a DNA sequence and the methylase is a DNA methylase.

14. The method of any one of claims 7 to 12 wherein the nucleotide sequence is a DNA sequence and the methylase is a DNA methylase.

15. The method of any one of claims 2, 5, 8 and 11 wherein the host cell is selected from the group consisting of a bacterial cell, a yeast cell and an insect cell.

16. The method of claim 15 wherein step (i) comprises transforming the host cell with a first recombinant vector comprising the selected nucleotide sequence and a second recombinant vector comprising the nucleotide sequence encoding a methylase enzyme.

17. The method of claim 15 wherein step (i) comprises transforming the host cell with a recombinant vector comprising the selected nucleotide sequence and transforming the host cell with a nucleotide sequence encoding a methylase enzyme by inserting the methylase enzyme-encoding nucleotide sequence into the genome of the host cell.

18. The method of claim 16 or 17 wherein the recombinant vectors are selected from the group consisting of plasmids, cosmids, fosmids, BAC's and YAC's.

19. The method of claim 18 wherein the recombinant vector is a plasmid selected from the group consisting of pUC8, pUC19, pBR322, pBluescript and pSP64.

20. The method of claim 13 or 14 wherein the methylase enzyme is a mammalian DNA methylase enzyme.

21. The method of claim 20 wherein the methylase enzyme is selected from the group consisting of DNMT1 , DNMT3a, and DNMT3b.

22. The method of claim 13 or 14 wherein the methylase enzyme is a non- mammalian methylase enzyme.

23. The method of claim 22 wherein the methylase enzyme is Sssl methylase and the host cell is E. coli.

24. The method of claim 1 wherein the unstable nucleotide sequence is a portion of or all of the HIV genome.

25. The method of any one of claims 3, 6, 9 and 12 wherein the host cell is a mammalian cell.

26. The method of any one of claims 3, 6, 9 and 12 wherein the host cell is a plant cell.

27. The method of claim 25 wherein the mammalian cell is selected from the group consisting of a CHO cell and an African green monkey kidney cell.