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Fragile Sites—Cytogenetic Similarity with Molecular Diversity

1999; Elsevier BV; Volume: 64; Issue: 2 Linguagem: Inglês

10.1086/302267

1537-6605

Grant R. Sutherland, Robert I. Richards,

Genomics and Rare Diseases

When examined in metaphase chromosome preparations, all fragile sites appear as a gap or discontinuity in chromosome structure. These gaps, which are induced by several specific treatments of cultured cells, are of variable width and promote chromosome breakage with variable efficiency. Within a single metaphase, it is not possible to distinguish between a fragile site and random chromosomal damage. Only the statistically significant recurrence of a lesion at the same locus and under the same culture conditions delineates fragile sites. Several classes of fragile sites have now been characterized at the molecular level. The “rare” fragile sites contain tandemly repeated sequences of varying complexity, which undergo expansions or, occasionally, contractions. The common fragile sites remain as enigmatic at the molecular level as the rare ones once were at the genetic level (Sutherland Sutherland, 1985Sutherland GR The enigma of the fragile X chromosome.Trends Genet. 1985; 1: 108-112Abstract Full Text PDF Scopus (23) Google Scholar). The cytogenetic expression of a fragile site is but one manifestation of genomic instability that is generated by the DNA sequences at fragile-site loci. For instance, the rare folate-sensitive fragile sites are associated with transcriptional silencing of genes, and some of the common fragile sites may also affect gene expression by creating local regions of chromosomal instability. The current challenge is to understand the mechanisms of this instability and its biological significance. Fragile sites are classified according to their frequency and the conditions or agents that induce their expression (Sutherland et al. Sutherland et al., 1996Sutherland GR Baker E Richards RI Fragile sites.Encyclopedia Molec Biol Molec Med. 1996; 2: 313-318Google Scholar). Common fragile sites appear to be part of normal chromosomal structure and are probably present at all common fragile-site loci in all individuals. Rare fragile sites vary in frequency from only a handful of case reports up to 1 in 40 chromosomes. A classification of fragile sites and some details of their cytogenetic and molecular properties are given in table 1.Table 1Classification and Some Properties of Fragile SitesClassInduced byNumber RecognizedNumber ClonedRepeat MotifRare: 1. Folate sensitiveLow folate and thymidine, FUdR, MTX, high thymidine235CCG 2. Distamycin A induciblea. Distamycin A, other minor groove-binding oligopeptide antibiotics, and BrdU2133 bp AT-richb. As above, but not BrdU (recorded only in the Japanese)30… 3. BrdU inducibleBrdU or BrdC21∼42 bp variable AT-richCommon: 4. Aphidicolin inducibleAphidicolin for at least 24 h763None obvious 5. 5-Azacytidine inducible5-Azacytidine for no more than 8 h40… 6. BrdU inducibleBrdU for no more than 8 h60… 7. Adenovirus 12 inducibleAdenovirus 12 in U1 small nuclear RNA genes in p53-expressing cells40… Open table in a new tab Fragile sites are identifiable as gaps or chromosomal breaks in only a fraction of metaphase spreads from a given individual. At one extreme is FRA16B, which, when induced by berenil, may be found in >90% of metaphases (Schmid et al. Schmid et al., 1986Schmid M Feichtinger W Jessberger A Köhler J Lange R The fragile site (16)(q22). I. Induction by AT-specific DNA-ligands and population frequency.Hum Genet. 1986; 74: 67-73PubMed Google Scholar). At the opposite end of the spectrum are the common aphidicolin fragile sites. Even the most conspicuous of these, FRA3B, is rarely seen in >10% of metaphases (Smeets et al. Smeets et al., 1986Smeets DFCM Scheres JMJC Hustinx TWJ The most common fragile site in man is 3p14.Hum Genet. 1986; 72: 215-220Crossref PubMed Scopus (96) Google Scholar), and many of the other common fragile sites are seen in <5% of metaphases. Whatever the mechanisms are that result in fragile-site expression, they usually operate successfully in only a minority of cells. Most of the treatments that induce fragile sites (e.g., perturbed nucleotide pools and aphidicolin) result in slowing DNA replication, particularly at fragile-site loci (Hansen et al. Hansen et al., 1997Hansen RS Canfield TK Fjeld AD Mumm S Laird CD Gartler SM A variable domain of delayed replication in FRAXA fragile X chromosomes: X inactivation-like spread of late replication.Proc Natl Acad Sci USA. 1997; 94: 4587-4592Crossref PubMed Scopus (83) Google Scholar; Le Beau et al. Le Beau et al., 1998Le Beau MM Rassool FV Neilly ME Espinosa III, R Glover TW Smith DI McKeithan TW Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: implications for the mechanism of fragile site induction.Hum Mol Genet. 1998; 7: 755-761Crossref PubMed Scopus (180) Google Scholar). It is thus possible that the common fragile sites, and perhaps the rare ones, arise because of incompletely replicated DNA sequences that do not package completely for mitosis. Whereas such packaging is completed in most cells, in a varying, often small proportion of cells it may not be completed before the end of G2, and it is in these cells that fragile sites manifest themselves as gaps or breaks. Only three of the different classes of rare fragile sites have been studied at the molecular level. Most is known about the rare folate-sensitive fragile sites, of which five members have been cloned. This group includes FRAXA, the site of the molecular lesion in the fragile X syndrome gene FMR1, which underlies the most common familial form of mental retardation. The normal alleles at the FRAXA locus vary in size from ∼6–55 CCG repeats, mostly with interspersed CCT units after every 9–10 CCG units (Hirst et al. Hirst et al., 1994Hirst MC Grewal PK Davies KE Precursor arrays for triplet repeat expansion at the fragile X locus.Hum Mol Genet. 1994; 3: 1553-1560Crossref PubMed Scopus (111) Google Scholar), although there is variation in this pattern, with some alleles having no or only one CCT unit. In Tunisian Jews, there is an increased incidence of normal alleles without CCT units, and this is likely to be the basis of an apparently high incidence of fragile X syndrome in this ethnic group (Falik-Zaccai et al. Falik-Zaccai et al., 1997Falik-Zaccai TC Shachak E Yalon M Lis Z Borochowitz Z Macpherson JN Nelson DL et al.Predisposition to the fragile X syndrome in Jews of Tunisian descent is due to the absence of AGG interruptions on a rare Mediterranean haplotype.Am J Hum Genet. 1997; 60: 103-112PubMed Google Scholar). The increased length of premutation and mutation alleles appears to be due to increased numbers of CCG units alone. Furthermore, the alleles that expand to give pre- and full mutations are in linkage disequilibrium with microsatellite (Richards et al. Richards et al., 1992Richards RI Holman K Friend K Kremer E Hillen D Staples A Brown WT et al.Evidence of founder chromosomes in fragile X syndrome.Nat Genet. 1992; 1: 257-260Crossref PubMed Scopus (146) Google Scholar) and single nucleotide polymorphisms (SNPs; Gunter et al. Gunter et al., 1998Gunter C Paradee W Crawford DC Meadows KA Newman J Kunst CB Nelson DL et al.Re-examination of factors associated with expansion of CGG repeats using a single nucleotide polymorphism in FMR1.Hum Mol Genet. 1998; 7: 1935-1946Crossref PubMed Scopus (57) Google Scholar) within the gene or close to its 5′ end. The presence of one of these SNPs, ATL1, can be used to predict which alleles are likely to proceed to expansion (Gunter et al. Gunter et al., 1998Gunter C Paradee W Crawford DC Meadows KA Newman J Kunst CB Nelson DL et al.Re-examination of factors associated with expansion of CGG repeats using a single nucleotide polymorphism in FMR1.Hum Mol Genet. 1998; 7: 1935-1946Crossref PubMed Scopus (57) Google Scholar), although it is unlikely that the ATL1 polymorphism, in the first intron of FMR1, has functional significance in relation to expansion. More likely, it is in linkage disequilibrium with some CCG repeat structure that promotes expansion. The only other folate-sensitive fragile site for which there is information on allele structure is FRA16A (Nancarrow et al. Nancarrow et al., 1995Nancarrow JK Holman K Mangelsdorf M Hori T Denton M Sutherland GR Richards RI Molecular basis of p(CCG)n repeat instability at the FRA16A fragile site locus.Hum Mol Genet. 1995; 4: 367-372Crossref PubMed Scopus (34) Google Scholar). As with FRAXA, some of the longer alleles that lack CCT interruption might be expected to undergo expansion more readily than interrupted alleles. However, too few expanded alleles are available for study to test this prediction rigorously. The cloning of FRA16B showed that repeat sequences other than trinucleotides could undergo expansion and result in a fragile site (Yu et al. Yu et al., 1997Yu S Mangelsdorf M Hewett D Hobson L Baker E Eyre H Lapsys N et al.Human chromosomal fragile site FRA16B is an amplified AT-rich minisatellite repeat.Cell. 1997; 88: 367-374Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). At this locus, a 33-bp AT-rich repeat, which is highly polymorphic on normal chromosomes, expands up to several thousand copies to yield a fragile site. Such large expansions can be viewed only on pulse-field gels, where variation in size by a relatively small number of copies would not be resolved. Hence, although the expansions in different families with FRA16B are clearly distinct, neither size variation within families nor somatic instability in carriers of this fragile site has been observed. Fragile site FRA10B differs from FRA16B in that, whereas the former is induced only by nucleotide analogues such as BrdU or BrdC, the latter is also (indeed, more strongly) induced by DNA minor groove-binding agents such as distamycin A and berenil. FRA10B is the most complex fragile site yet studied. It is an expansion of 5–100 kb, consisting of an AT-rich minisatellite repeat unit of ∼42 bp in length, which varies in size and composition between families (Hewett et al. Hewett et al., 1998Hewett DR Handt O Hobson L Mangelsdorf M Eyre H Baker E Sutherland GR et al.FRA10B structure reveals common elements in repeat expansion and chromosomal fragile site genesis.Mol Cell. 1998; 1: 773-781Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The expanded sequence shows both somatic and intergenerational instability. Three common fragile sites (FRA3B, FRA7G, and FRA7H) have also been studied in some detail (Mishmar et al. Mishmar et al., 1998Mishmar D Rahat A Scherer SW Nyakatura G Hinzmann B Kohwi Y Mandel-Gutfroind Y et al.Molecular characterization of a common fragile site (FRA7H) on human chromosome 7 by the cloning of a simian virus 40 integration site.Proc Natl Acad Sci USA. 1998; 14: 8141-8146Crossref Scopus (191) Google Scholar). In contrast to the rare fragile sites, here the fragility occurs over a region of tens to hundreds of kilobases rather than at a single point. Sequence analysis of these regions reveals no striking features that could account for the fragile site, although there are regions of decreased stability and increased flexibility of the DNA that might be significant (Mishmar et al. Mishmar et al., 1998Mishmar D Rahat A Scherer SW Nyakatura G Hinzmann B Kohwi Y Mandel-Gutfroind Y et al.Molecular characterization of a common fragile site (FRA7H) on human chromosome 7 by the cloning of a simian virus 40 integration site.Proc Natl Acad Sci USA. 1998; 14: 8141-8146Crossref Scopus (191) Google Scholar; Palin et al. Palin et al., 1998Palin AH Critcher R Fitzgerald DJ Anderson JN Farr CJ Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility.J Cell Sci. 1998; 111: 1623-1634PubMed Google Scholar). FRA3B occurs within the FHIT gene, but the other two fragile sites are not known to be within genes. These common fragile sites are late-replicating regions, and it is of interest that aphidicolin, which induces these fragile sites, further delays their replication (Le Beau et al. Le Beau et al., 1998Le Beau MM Rassool FV Neilly ME Espinosa III, R Glover TW Smith DI McKeithan TW Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: implications for the mechanism of fragile site induction.Hum Mol Genet. 1998; 7: 755-761Crossref PubMed Scopus (180) Google Scholar). This finding supports the model that these fragile sites may simply represent very late–replicating regions of DNA, which, in a minority of cells, fail to complete replication before mitosis begins and, for this reason, do not package well into chromosomes. A common aphidicolin type fragile site in the Chinese hamster has been cloned on the basis of its increased sensitivity for the incorporation of a linearized plasmid containing a selectable drug-resistance marker. Sequence analysis showed the region of the fragile site to be AT-rich, with a number of other features, including homologies to yeast autonomous replicating sequences and a consensus sequence for replication origins (Palin et al. Palin et al., 1998Palin AH Critcher R Fitzgerald DJ Anderson JN Farr CJ Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility.J Cell Sci. 1998; 111: 1623-1634PubMed Google Scholar). Fragile sites inducible by adenovirus 12 occur in the small nuclear RNA gene clusters but only in cells expressing p53. It has been postulated that a viral protein causes p53 to undergo a gain of function, leading either to perturbed transcription by RNA polymerase II or to inefficient chromatin condensation. As Li et al. (Li et al., 1998Li Z Bailey AD Buchowski J Weiner AM A tandem array of minimal U1 small nuclear RNA genes is sufficient to generate a new adenovirus type 12-inducible chromosome fragile site.J Virol. 1998; 72: 4205-4211PubMed Google Scholar) note, continued replication or transcription beyond G2 might be expected to interfere with chromatin compaction. Although fragile sites are themselves visual manifestations of genome instability, there is considerable evidence that genome instability at these loci occurs at other levels. Because most or all of the rare fragile sites are prone to intergenerational and somatic repeat expansion, it is worth considering how this instability comes about. In the case of FRAXA, the best-known example, certain normal alleles can increase in size from within the normal range of copy numbers into the premutation range. Mechanisms to account for instability include unequal crossing over, gene conversion, and replication slippage (see Sinden Sinden, 1999Sinden RR Biological implications of the DNA structures associated with disease-causing triplet repeats.Am J Hum Genet. 1999; 64 (in this issue): 346-353Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar [in this issue]). These events are apparently rare, and the rate at which they occur is probably little different from the mutation rate of other simple tandem repeats (∼10−3 per locus/generation). Once the number of uninterrupted repeats exceeds ∼80, the length of an Okazaki fragment, replication slippage is likely to be the predominant mechanism by which increasingly larger size changes occur, the process of dynamic mutation (Richards and Sutherland Richards and Sutherland, 1994Richards RI Sutherland GR Simple repeat DNA is not replicated simply.Nat Genet. 1994; 6: 114-116Crossref PubMed Scopus (288) Google Scholar; Sutherland et al. Sutherland et al., 1998Sutherland GR Baker E Richards RI Fragile sites still breaking.Trends Genet. 1998; 14: 501-506Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This model has recently received experimental support (Freudenreich et al. Freudenreich et al., 1998Freudenreich CH Kantrow SM Zakian VA Expansion and length-dependent fragility of CTG repeats in yeast.Science. 1998; 279: 853-856Crossref PubMed Scopus (357) Google Scholar; Sarkar et al. Sarkar et al., 1998Sarkar PS Chang HC Boudi FB Reddy S CTG repeats show bimodal amplification in E. coli.Cell. 1998; 95: 531-540Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Superimposed on this process are a number of factors such as the age and sex of the individual in which the changes occur. The most likely series of events in the genesis of an individual with a FRAXA full mutation is shown in figure 1. An ovum with a full mutation is produced (by either a pre- or a full mutation carrier) and fertilized. This full mutation is unmethylated (Malter et al. Malter et al., 1997Malter HE Iber JC Willemsen R de Graaff E Tarleton JC Leisti J Warren ST et al.Characterization of the full fragile X syndrome mutation in fetal gametes.Nat Genet. 1997; 15: 165-169Crossref PubMed Scopus (164) Google Scholar), and it remains so throughout early embryogenesis. The unmethylated mutation is somatically unstable and gives rise to cell lineages with different numbers of copies of the repeating unit. Most of these are probably breakdown products with fewer copies of the repeat than are found in the ovum, but some may contain additional copies. Once the expanded repeat undergoes CpG methylation, further changes in repeat copy number are rare. Methylation is probably not complete until 12–14 weeks of gestation. At least in the case of the FMR1 gene, methylation also leads to transcriptional silencing of the allele on the fragile X chromosome. In the male fetus, there is premeiotic selection against germ cells that contain the full mutation in favor of cells with a premutation (Ashley-Koch et al. Ashley-Koch et al., 1998Ashley-Koch AE Robinson H Glicksman AE Nolin SL Schwartz CE Brown WT Turner G et al.Examination of factors associated with instability of the FMR1 CGG repeat.Am J Hum Genet. 1998; 63: 776-785Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Eventually spermatogenesis results in sperm with premutations only. However, no selection occurs in the ovary of a female fetus, so full mutations persist in the female germline. During oogenesis, when CpG methylation is erased, instability is restored to the CCG repeat, and ova with different-sized mutations are generated. Finally, as individuals age, there is very slow selection against somatic cells with the largest alleles (Ashley-Koch et al. Ashley-Koch et al., 1998Ashley-Koch AE Robinson H Glicksman AE Nolin SL Schwartz CE Brown WT Turner G et al.Examination of factors associated with instability of the FMR1 CGG repeat.Am J Hum Genet. 1998; 63: 776-785Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In females, there is slow selection against cells in which the fragile site is on the active X chromosome (Rousseau et al. Rousseau et al., 1991Rousseau F Heitz D Oberlé I Mandel J-L Selection in blood cells from female carriers of the fragile X syndrome: inverse correlation between age and proportion of active X chromosomes carrying the full mutation.J Med Genet. 1991; 28: 830-836Crossref PubMed Scopus (91) Google Scholar). It is likely that this series of events is also applicable to FRAXE but not entirely to the autosomal sites. Thus, full mutations in FRA16A do not appear to be subject to selection in male germ cells (Nancarrow et al. Nancarrow et al., 1994Nancarrow JK Kremer E Holman K Eyre H Doggett NA Le Paslier D Callen DF et al.Implications of FRA16A structure for the mechanism of chromosomal fragile site genesis.Science. 1994; 264: 1938-1941Crossref PubMed Scopus (132) Google Scholar). Although intergenerational and somatic instability can occur at FRA10B and probably also at FRA16B, the rates at which these changes occur are much lower than at folate-sensitive fragile sites, perhaps because replication slippage is less likely with longer-repeat motifs. Other mechanisms, such as gene conversion, may account for changes in allele size at FRA10B and FRA16B (Jeffreys et al. Jeffreys et al., 1994Jeffreys A Tamaki K MacLeod A Monckton DG Neil DL Armour JAL Complex gene conversion events in germline mutation at minisatellites.Nat Genet. 1994; 6: 136-145Crossref PubMed Scopus (452) Google Scholar). Genomic instability at the common fragile sites has been documented extensively for FRA3B and to a lesser extent for FRA7G (Huang et al. Huang et al., 1998Huang H Qian C Jenkins RB Smith DI Fish mapping of YAC clones at human chromosomal band 7q31.2: identification of YACs spanning FRA7G within the common region of LOH in breast and prostate cancer.Genes Chromosom Cancer. 1998; 21: 152-159Crossref PubMed Scopus (68) Google Scholar). Instability at FRA3B occurs by recombination between long L1 sequences (Inoue et al. Inoue et al., 1997Inoue H Ishii H Alder H Snyder E Druck T Huebner K Croce CM Sequence of the FRA3B common fragile region: implications for the mechanism of FHIT deletion.Proc Natl Acad Sci USA. 1997; 94: 14584-14589Crossref PubMed Scopus (118) Google Scholar) and generates deletions in the FHIT gene in a range of solid tumors. FRA7G is within an area of instability in prostate cancers, but the mechanism of its instability has not been determined. Other manifestations of genomic instability at FRA3B include its function as a target for viral integration, specifically of HPV16. There is also evidence for the common fragile sites being involved in gene amplification by being points of breakage in a breakage-fusion bridge mechanism (Coquelle et al. Coquelle et al., 1998Coquelle A Toledo F Stern S Bieth A Debatisse M A new role for hypoxia in tumor progression: induction of fragile site triggering genomic rearrangements and formation of complex DMs and HSRs.Mol Cell. 1998; 2: 259-265Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The biological significance of chromosomal fragility is not easily generalized among fragile loci. Two of the rare fragile sites on the X chromosome are manifestations of disease (mental retardation)–producing dynamic mutations, whereas the third rare fragile site on this chromosome, FRAXF, is not found within any known gene and does not lead to any obvious phenotypic abnormality. The autosomal fragile sites of this type may be associated with transcriptional silencing of genes in which they might be located. Breakage at fragile sites may be capable of producing chromosomal deletion syndromes, as perhaps occurs when FRA11B breakage leads to Jacobsen syndrome (Jones et al. Jones et al., 1995Jones C Penny L Mattina T Yu S Baker E Voullaire L Langdon WY et al.Association of a chromosome deletion syndrome with a fragile site within the proto-oncogene CBL2.Nature. 1995; 376: 145-149Crossref PubMed Scopus (170) Google Scholar). It is of interest that none of the rare folate-sensitive fragile sites has been identified in species other than humans, yet repeats that are potentially capable of expansion to fragile sites have been identified in other species. Fragile sites are probably all associated with localized genomic instability; although this instability might lead only to the “gap” seen in cytogenetic preparations, it is also possible that the consequences are more significant. There is growing evidence that some common fragile sites predispose their surrounding region to the localized chromosomal instability seen in certain cancers. FRA3B is one such region of instability, and abnormal transcripts of the FHIT gene in which it is located are found in a range of tumor and normal cell types (Siprashvili et al. Siprashvili et al., 1997Siprashvili Z Sozzi G Barnes LD McCue P Robinson AK Eryomin V Sard L et al.Replacement of FHIT in cancer cells suppresses tumorigenicity.Proc Natl Acad Sci USA. 1997; 94: 13771-13776Crossref PubMed Scopus (350) Google Scholar; Carapeti et al. Carapeti et al., 1998Carapeti M Aguiar RC Sill H Goldman JM Cross NC Aberrant transcripts of the FHIT gene are expressed in normal and leukaemic haemopoietic cells.Br J Cancer. 1998; 78: 601-605Crossref PubMed Scopus (14) Google Scholar; Otterson et al. Otterson et al., 1998Otterson GA Xiao GH Geradts J Jin F Chen WD Niklinska W Kaye FJ et al.Protein expression and functional analysis of the FHIT gene in human tumor cells.J Natl Cancer Inst. 1998; 18: 426-432Crossref Scopus (76) Google Scholar). The human caveolin genes CAV1 and CAV2 are located in the vicinity of FRA7G, which is frequently deleted in human cancers (Engelman et al. Engelman et al., 1998bEngelman JA Zhang XL Lisanti MP Genes encoding human caveolin-1 and -2 are co-localized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers.FEBS Lett. 1998b; 436: 403-410Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Caveolin-1 has been shown to have a role in the anchorage-dependent inhibition of growth in NIH 3T3 cells (Galbiati et al. Galbiati et al., 1998Galbiati F Volonte D Engelman JA Watanabe G Burk R Pestell RG Lisanti MP Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade.EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (425) Google Scholar). The caveolins are therefore candidates for the tumor-suppressor gene presumed to be located in the FRA7G region (Engelman et al. Engelman et al., 1998aEngelman JA Zhang X Galbiati F Volonte D Sotgia F Pestell RG Minetti C et al.Molecular genetics of the calveolin gene family: implications for human cancers, diabetes, Alzheimer disease, and muscular dystrophy.Am J Hum Genet. 1998a; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). By similar mechanisms, fragile sites may also lead to gene amplification in tumors. The noncompacted state of the DNA at fragile sites may also facilitate the integration of viruses into the genome.