Synthesis-dependent Strand Annealing Pathway Research Articles

Mitotic Gene Conversion Tracts Associated with Repair of a Defined Double-Strand Break in Saccharomyces cerevisiae.

Mitotic recombination between homologous chromosomes leads to the uncovering of recessive alleles through loss of heterozygosity. In the current study, a defined double-strand break was used to initiate reciprocal loss of heterozygosity between diverged homologs of chromosome IV in Saccharomyces cerevisiae These events resulted from the repair of two broken chromatids, one of which was repaired as a crossover and the other as a noncrossover. Associated gene conversion tracts resulting from the donor-directed repair of mismatches formed during strand exchange (heteroduplex DNA) were mapped using microarrays. Gene conversion tracts associated with individual crossover and noncrossover events were similar in size and position, with half of the tracts being unidirectional and mapping to only one side of the initiating break. Among crossover events, this likely reflected gene conversion on only one side of the break, with restoration-type repair occurring on the other side. For noncrossover events, an ectopic system was used to directly compare gene conversion tracts produced in a wild-type strain to heteroduplex DNA tracts generated in the absence of the Mlh1 mismatch-repair protein. There was a strong bias for unidirectional tracts in the absence, but not in the presence, of Mlh1 This suggests that mismatch repair acts on heteroduplex DNA that is only transiently present in noncrossover intermediates of the synthesis dependent strand annealing pathway. Although the molecular features of events associated with loss of heterozygosity generally agreed with those predicted by current recombination models, there were unexpected complexities in associated gene conversion tracts.

Double-Strand Break Repair Assays Determine Pathway Choice and Structure of Gene Conversion Events inDrosophila melanogaster

Double-strand breaks (DSBs) must be accurately and efficiently repaired to maintain genome integrity. Depending on the organism receiving the break, the genomic location of the DSB, and the cell-cycle phase in which it occurs, a DSB can be repaired by homologous recombination (HR), nonhomologous end-joining (NHEJ), or single-strand annealing (SSA). Two novel DSB repair assays were developed to determine the contributions of these repair pathways and to finely resolve repair event structures in Drosophila melanogaster. Rad51-dependent homologous recombination is the preferred DSB repair pathway in mitotically dividing cells, and the pathway choice between HR and SSA occurs after end resection and before Rad51-dependent strand invasion. HR events are associated with long gene conversion tracts and are both bidirectional and unidirectional, consistent with repair via the synthesis-dependent strand annealing pathway. Additionally, HR between diverged sequences is suppressed in Drosophila, similar to levels reported in human cells. Junction analyses of rare NHEJ events reveal that canonical NHEJ is utilized in this system.

Unwinding of synthetic replication and recombination substrates by Srs2

The budding yeast Srs2 protein possesses 3′ to 5′ DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).

Double-strand break repair in bacteriophage T4: recombination effects of 3'-5' exonuclease mutations.

The role of 3'-5' exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i x ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3'-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (<100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesis-dependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed.

Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.

Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing.

Bloom syndrome, characterized by a predisposition to cancer, is caused by mutation of the RecQ DNA helicase gene BLM. The precise function of BLM remains unclear. Previous research suggested that Drosophila BLM functions in the repair of DNA double-strand breaks. Most double-strand breaks in flies are repaired by homologous recombination through the synthesis-dependent strand-annealing pathway. Here, we demonstrate that Drosophila BLM mutants are severely impaired in their ability to carry out repair DNA synthesis during synthesis-dependent strand annealing. Consequently, repair in the mutants is completed by error-prone pathways that create large deletions. These results suggest a model in which BLM maintains genomic stability by promoting efficient repair DNA synthesis and thereby prevents double-strand break repair by less precise pathways.

Abortive gap repair: underlying mechanism for Ds element formation.

The mechanism by which the maize autonomous Ac transposable element gives rise to nonautonomous Ds elements is largely unknown. Sequence analysis of native maize Ds elements indicates a complex chimeric structure formed through deletions of Ac sequences with or without insertions of Ac-unrelated sequence blocks. These blocks are often flanked by short stretches of reshuffled and duplicated Ac sequences. To better understand the mechanism leading to Ds formation, we designed an assay for detecting alterations in Ac using transgenic tobacco plants carrying a single copy of Ac. We found frequent de novo alterations in Ac which were excision rather than sequence dependent, occurring within Ac but not within an almost identical Ds element and not within a stable transposase-producing gene. The de novo DNA rearrangements consisted of internal deletions with breakpoints usually occurring at short repeats and, in some cases, of duplication of Ac sequences or insertion of Ac-unrelated fragments. The ancient maize Ds elements and the young Ds elements in transgenic tobacco showed similar rearrangements, suggesting that Ac-Ds elements evolve rapidly, more so than stable genes, through deletions, duplications, and reshuffling of their own sequences and through capturing of unrelated sequences. The data presented here suggest that abortive Ac-induced gap repair, through the synthesis-dependent strand-annealing pathway, is the underlying mechanism for Ds element formation.