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M Jasin

Publications and source records attributed to M Jasin.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange.

Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

Animals↗
PubMed2000

Chromosome breaks and genomic instability.

Tumorigenesis is known to result from multiple genetic changes. Although endogenous and environmental insults can damage DNA, cellular mechanisms exist to repair various forms of damage or to kill those cells irreparably damaged. Hence, the accumulation of numerous genetic changes that would lead to cancer in normal cells is extremely rare. Nevertheless, disruption of a DNA repair pathway has the potential to expedite tumorigenesis by resulting in a cell that is hypermutable. Multiple pathways exist to repair the various forms of DNA damage that can cause mutagenesis. Recent studies have demonstrated a key role for homologous recombination in DNA repair, in particular in the repair chromosomal double-strand breaks. This review summarizes those studies and discusses how disruption of homologous recombination pathways can create genetic instability.

Animals↗
PubMed1999

A mouse homolog of the Saccharomyces cerevisiae meiotic recombination DNA transesterase Spo11p.

The Saccharomyces cerevisiae Spo11 protein is thought to catalyze formation of the DNA double-strand breaks that initiate meiotic recombination. We have cloned cDNA and genomic DNA for a mouse gene encoding a protein with significant sequence similarity to conserved domains found in proteins of the Spo11p family. This putative mouse Spo11 gene maps to the distal region of chromosome 2 (homologous to human chromosome 20q13.2-q13.3) and comprises at least 12 exons, spanning approximately 15-18 kb. Strong expression of the Spo11 message is seen in juvenile and adult testis by RNA in situ hybridization, RT-PCR, and Northern blot, with much weaker expression in thymus and brain. In situ hybridization detects expression in oocytes of embryonic ovary, but not of adult ovary. RT-PCR and in situ hybridization analyses of a time course of juvenile testis development indicate that Spo11 expression begins in early meiotic Prophase I, prior to the pachytene stage, with increasing accumulation of mRNA through the pachytene stage. Taken together, these results strongly suggest that this gene encodes the functional homolog of yeast Spo11p, which in turn suggests that the mechanism of meiotic recombination initiation is conserved between yeast and mammals.

Amino Acid Sequence↗
PubMed1999

XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.

Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.

Animals↗
PubMed1999

Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination.

The repair of DNA double-strand breaks is essential for cells to maintain their genomic integrity. Two major mechanisms are responsible for repairing these breaks in mammalian cells, non-homologous end-joining (NHEJ) and homologous recombination (HR): the importance of the former in mammalian cells is well established, whereas the role of the latter is just emerging. Homologous recombination is presumably promoted by an evolutionarily conserved group of genes termed the Rad52 epistasis group. An essential component of the HR pathway is the strand-exchange protein, known as RecA in bacteria or Rad51 in yeast. Several mammalian genes have been implicated in repair by homologous recombination on the basis of their sequence homology to yeast Rad51: one of these is human XRCC2. Here we show that XRCC2 is essential for the efficient repair of DNA double-strand breaks by homologous recombination between sister chromatids. We find that hamster cells deficient in XRCC2 show more than a 100-fold decrease in HR induced by double-strand breaks compared with the parental cell line. This defect is corrected to almost wild-type levels by transient transfection with a plasmid expressing XRCC2. The repair defect in XRCC2 mutant cells appears to be restricted to recombinational repair because NHEJ is normal. We conclude that XRCC2 is involved in the repair of DNA double-strand breaks by homologous recombination.

Animals↗
PubMed1999

Chromosome healing in mouse embryonic stem cells.

The addition of new telomeres to the ends of broken chromosomes, termed chromosome healing, has been extensively studied in unicellular organisms; however, its role in the mammalian cell response to double-strand breaks is unknown. A system for analysis of chromosome healing, which involves the integration of plasmid sequences immediately adjacent to a telomere, has been established in mouse embryonic stem cells. This "marked" telomere contains a neo gene for positive selection in G418, an I-SceI endonuclease recognition sequence for introducing double-strand breaks, and a herpes simplex virus thymidine kinase gene for negative selection with ganciclovir for cells that have lost the telomere. Transient expression of the I-SceI endonuclease results in terminal deletions involving telomeric repeat sequences added directly onto the end of the broken chromosome. The sites of addition of the new telomeres contain short regions of complementarity to telomeric repeat sequences. The most common site of addition is the last A of the ATAA 3' overhang generated by the I-SceI endonuclease, without the loss of a single nucleotide from the end of the chromosome. The next most frequent site involved 5 bp of complementarity, which occurred after the loss of four nucleotides from the end of the chromosome. The new telomeres are generally much shorter than in the parental cell line, and most increase in size with time in culture. These results demonstrate that chromosome healing is a mechanism for repair of chromosome breaks in mammalian cells.

Animals↗
PubMed1999

Palindromic DNA and genome stability. Further studies.

Unusual DNA structures promote genetic instability. One such example is hairpin DNA, which can form from palindromic sequences and triplet repeats, and is also a characteristic intermediate in V(D)J recombination. We previously found that a large 15.3-kb palindrome that was introduced as a transgene into the mouse germline was highly unstable. Although it could be transmitted, the transgene was found to be rearranged in up to 56% of the progeny, and rearrangement events often involved deletion at the center of symmetry. Here, the fine structure of centrally deleted palindromes was sampled by analysis of recombinant junctions isolated from testes DNA, providing further evidence for a model, previously proposed, that accounts for such deletions on the basis of a hairpin-tip nicking activity. In addition to central deletions, gene conversion events were also elevated in the transgenic palindrome. We have now analyzed instability in two mouse sublines in which (as a result of inversion) the transgenic palindrome had been shortened to 4.2 kb. In these sublines, the transgene was still subject to both rearrangement and gene conversion events at a high frequency, similar to the original 15.3-kb palindrome. Recombination was not limited to the sequences constituting the inverted repeat, but was seen to include sequences lying outside the palindrome. As discussed, the salient feature in all of these observations, a high level of genetic change associated with palindromic DNA, underscores the significance of hairpin DNA and hairpin-tip nicking in genome stability.

Animals↗
PubMed1999

Brca1 controls homology-directed DNA repair.

Germline mutations in BRCA1 confer a high risk of breast and ovarian tumors. The role of BRCA1 in tumor suppression is not yet understood, but both transcription and repair functions have been ascribed. Evidence that BRCA1 is involved in DNA repair stems from its association with RAD51, a homolog of the yeast protein involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination. We report here that Brca1-deficient mouse embryonic stem cells have impaired repair of chromosomal DSBs by homologous recombination. The relative frequencies of homologous and nonhomologous DNA integration and DSB repair were also altered. The results demonstrate a caretaker role for BRCA1 in preserving genomic integrity by promoting homologous recombination and limiting mutagenic nonhomologous repair processes.

Animals↗
PubMed1998

Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations.

To directly determine whether recombinational repair of double-strand breaks (DSBs) can occur between heterologous chromosomes and lead to chromosomal rearrangements in mammalian cells, we employed an ES cell system to analyze recombination between repeats on heterologous chromosomes. We found that recombination is induced at least 1000-fold following the introduction of a DSB in one repeat. Most (98%) recombinants repaired the DSB by gene conversion in which a small amount of sequence information was transferred from the unbroken chromosome onto the broken chromosome. The remaining recombinants transferred a larger amount of information, but still no chromosomal aberrations were apparent. Thus, mammalian cells are capable of searching genome-wide for sequences that are suitable for DSB repair. The lack of crossover events that would have led to translocations supports a model in which recombination is coupled to replication.

Alleles↗
PubMed1998

Homology-directed repair is a major double-strand break repair pathway in mammalian cells.

Mammalian cells have been presumed to repair potentially lethal chromosomal double-strand breaks (DSBs) in large part by processes that do not require homology to the break site. This contrasts with Saccharomyces cerevisiae where the major DSB repair pathway is homologous recombination. Recently, it has been determined that DSBs in genomic DNA in mammalian cells can stimulate homologous recombination as much as 3 or 4 orders of magnitude, suggesting that homology-directed repair may play an important role in the repair of chromosomal breaks. To determine whether mammalian cells use recombinational repair at a significant level, we have analyzed the spectrum of repair events at a defined chromosomal break by using direct physical analysis of repair products. When an endonuclease-generated DSB is introduced into one of two direct repeats, homologous repair is found to account for 30-50% of observed repair events. Both noncrossover and deletional homologous repair products are detected, at approximately a 1:3 ratio. These results demonstrate the importance of homologous recombination in the repair of DSBs in mammalian cells. In the remaining observed repair events, DSBs are repaired by nonhomologous processes. The nonhomologous repair events generally result in small deletions or insertions at the break site, although a small fraction of events result in larger chromosomal rearrangements. Interestingly, in two insertions, GT repeats were integrated at one of the broken chromosome ends, suggesting that DSB repair can contribute to the spread of microsatellite sequences in mammalian genomes.

Animals↗
PubMed1998

Gene conversion tracts from double-strand break repair in mammalian cells.

Mammalian cells are able to repair chromosomal double-strand breaks (DSBs) both by homologous recombination and by mechanisms that require little or no homology. Although spontaneous homologous recombination is rare, DSBs will stimulate recombination by 2 to 3 orders of magnitude when homology is provided either from exogenous DNA in gene-targeting experiments or from a repeated chromosomal sequence. Using a gene-targeting assay in mouse embryonic stem cells, we now investigate the effect of heterology on recombinational repair of DSBs. Cells were cotransfected with an endonuclease expression plasmid to induce chromosomal DSBs and with substrates containing up to 1.2% heterology from which to repair the DSBs. We find that heterology decreases the efficiency of recombinational repair, with 1.2% sequence divergence resulting in an approximately sixfold reduction in recombination. Gene conversion tract lengths were examined in 80 recombinants. Relatively short gene conversion tracts were observed, with 80% of the recombinants having tracts of 58 bp or less. These results suggest that chromosome ends in mammalian cells are generally protected from extensive degradation prior to recombination. Gene conversion tracts that were long (up to 511 bp) were continuous, i.e., they contained an uninterrupted incorporation of the silent mutations. This continuity suggests that these long tracts arose from extensive degradation of the ends or from formation of heteroduplex DNA which is corrected with a strong bias in the direction of the unbroken strand.

Animals↗
PubMed1998

Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells.

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.

Amino Acid Sequence↗
PubMed1997

Loss of heterozygosity induced by a chromosomal double-strand break.

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.

Alleles↗
PubMed1997

Palindrome resolution and recombination in the mammalian germ line.

Genetic instability is promoted by unusual sequence arrangements and DNA structures. Hairpin DNA structures can form from palindromes and from triplet repeats, and they are also intermediates in V(D)J recombination. We have measured the genetic stability of a large palindrome which has the potential to form a one-stranded hairpin or a two-stranded cruciform structure and have analyzed recombinants at the molecular level. A palindrome of 15.3 kb introduced as a transgene was found to be transmitted at a normal Mendelian ratio in mice, in striking contrast to the profound instability of large palindromes in prokaryotic systems. In a significant number of progeny mice, however, the palindromic transgene is rearranged; between 15 and 56% of progeny contain rearrangements. Rearrangements within the palindromic repeat occur both by illegitimate and homologous, reciprocal recombination. Gene conversion within the transgene locus, as quantitated by a novel sperm fluorescence assay, is also elevated. Illegitimate events often take the form of an asymmetric deletion that eliminates the central symmetry of the palindrome. Such asymmetric transgene deletions, including those that maintain one complete half of the palindromic repeat, are stabilized so that they cannot undergo further illegitimate rearrangements, and they also exhibit reduced levels of gene conversion. By contrast, transgene rearrangements that maintain the central symmetry continue to be unstable. Based on the observed events, we propose that one mechanism promoting the instability of the palindrome may involve breaks generated at the hairpin structure by a hairpin-nicking activity, as previously detected in somatic cells. Because mammalian cells are capable of efficiently repairing chromosome breaks through nonhomologous processes, the resealing of such breaks introduces a stabilizing asymmetry at the center of the palindrome. We propose that the ability of mammalian cells to eliminate the perfect symmetry in a palindromic sequence may be an important DNA repair pathway, with implications regarding the metabolism of palindromic repeats, the mutability of quasipalindromic triplet repeats, and the early steps in gene amplification events.

Animals↗