Laboratory of Lorraine S. Symington, Ph.D
Professor of Microbiology & Immunology
Resolution of Recombination Intermediates

DSB-induced HR initiates by the 5' to 3' degradation of the broken DNA ends to create 3' single-stranded DNA (ssDNA) tails. Rad51 binds to the resulting ssDNA tails to initiate pairing and strand invasion with homologous duplex DNA forming a displacement loop (D-loop). The 3' end from the broken chromosome is used to prime leading strand DNA synthesis templated by the donor duplex. By the canonical double-strand break repair (DSBR) model, the other end of the break interacts with the D-loop from the donor duplex to prime DNA synthesis and seal the break. The resulting double Holliday junction (dHJ) intermediate can be resolved to generate crossover (CO) or non-crossover (NCO) products. Intermediates containing a dHJ have been detected physically during mitotic and meiotic DSBR. During synthesis-dependent strand annealing (SDSA), the invading strand that has been extended by DNA synthesis is displaced and anneals to complementary sequences exposed by 5'-3' resection of the other side of the break yielding exclusively NCO products. The remaining gaps can be filled by DNA synthesis and the nicks ligated. For BIR a processive replication fork is established after strand invasion and DNA synthesis proceeds to the end of the chromosome. Use of the BIR pathway to repair a DSB from a non-sister chromatid results in LOH from the site of strand invasion to the telomere, and generates non-reciprocal translocations when it occurs between dispersed repeats. BIR is suppressed at DSBs, but plays an important role in telomere maintenance in the absence of telomerase.


resolution



Use of the SDSA mechanism in mitotic cells prevents crossovers and several helicases are proposed to unwind D-loops or inhibit second end capture. These include Srs2, Mph1/FANCM and RTEL1 and mutation of the gene encoding any one of them results in increased levels of crossovers associated with gene conversion. The dHJ intermediate formed by the DSBR mechanism must be dissolved or resolved to yield separate intact duplex molecules. Dissolution of dHJ intermediates requires the combined activity of the BLM/Sgs1 helicase, which drives migration of the constrained Holliday junctions, and the Top3-Rmi1 complex, which decatenates the interlinked strands between the two Holliday junctions eventually leading to non-crossover products. On the other hand, resolution through nucleolytic cleavage of the Holliday junctions can yield crossover (cutting inner strands of one Holliday junction and outer strands of the other) or non-crossover products (cutting both junctions in the same plane). Several proteins, including Mus81-Mms4/EME1, Slx1-Slx4 and Yen1/GEN1, able to cut HJs in vitro have been identified as candidate resolvases in yeast and mammals. Our recently published data show Mus81-Mms4 is the primary resolvase for DSB-induced mitotic crossovers in budding yeast with Yen1 serving a back-up function. Surprisingly, BIR was increased instead of the anticipated increase in non-crossover products in the mus81 yen1 mutant. Consistent with resolution of recombination intermediates being required for chromosome segregation, a large increase in spontaneous chromosome aneuploidy was found for the mus81 yen1 double mutant.



detection



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