Dynamic Processing of Displacement Loops during Recombinational DNA Repair

Abstract

Highlights d Development of a physical assay for D-loop detection in cells d Srs2, Mph1, and Sgs1-Top3-Rmi1 (STR) regulate D-loop levels in vivo d Two distinct pathways (Srs2 and Mph1, STR) target different D-loop species d Rdh54 delineates the two D-loop reversal pathways

Resolvases, Dissolvases, and Helicases in Homologous Recombination: Clearing the Road for Chromosome Segregation

“…This study also led to the conclusion that, in addition to D-loops, Sgs1, and Srs2, but not Mph1, also disassemble HJ-containing intermediates [59]. In vitro assays of D-loop reconstitution showed that Mph1 is extremely efficient in displacing these structures [60], a feature that supports a role for the helicase in promoting SDSA in vivo [13,61]. This Mph1-dependent bias to SDSA is fundamental to avoid the processing of recombination intermediates by nucleases that can contribute to the generation of COs (Figure 1).…”

“…Notably, Sgs1 is dispensable for full-length meiotic DSB resection, which mainly relies on Exo1 and Tel1 activities [75]. On the other hand, the STR complex, via Top3 decatenation activity, is capable of dismantling D-loops in vivo, favoring the formation of NCOs by the SDSA repair pathway both in the meiotic [76,77] and mitotic [13] programs ( Figure 1). Although in vitro assays using artificial D-loops suggested that Top3 preferentially dismantles protein-free D-loops [78,79], more recent single-molecule imaging analysis has revealed that Sgs1 possesses the ability to disrupt Rad51-bound ssDNA nucleofilaments [80].…”

“…Independently of the DSB repair program used, the Rad51/Dmc1 nucleofilament has the ability to test homology with an intact DNA donor sequence and promote strand exchange, leading to the formation of the displacement loop (D-loop) ( Figure 1). D-loop formation is subjected to dynamic reversibility, a process that has been proposed to contribute to the fidelity of HR [13]. The invading strand of the D-loop is extended by DNA polymerases that copy the lost information at the break site.…”

Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family

“…Rdh54 is important for HR in diploid cells, but roles in haploid cells are also emerging. Notably, the recently identified role for Rdh54 in regulating D-loop formation [174], and thereby HR levels, could become increasingly important in the absence of Rrm3 when greater numbers of HR substrates are likely to form at replication pause sites. That Sgs1-Top3-Rmi1 and Srs2 define two independent pathways of D-loop reversal could contribute to the synthetic lethality of the rrm3∆ mutation with sgs1∆ and srs2∆ mutations as well as its suppression by RAD51 deletion [62,83,173,174].…”

“…Notably, the recently identified role for Rdh54 in regulating D-loop formation [174], and thereby HR levels, could become increasingly important in the absence of Rrm3 when greater numbers of HR substrates are likely to form at replication pause sites. That Sgs1-Top3-Rmi1 and Srs2 define two independent pathways of D-loop reversal could contribute to the synthetic lethality of the rrm3∆ mutation with sgs1∆ and srs2∆ mutations as well as its suppression by RAD51 deletion [62,83,173,174]. Upregulation of Mph1, which also functions in D-loop disassembly [175], was also observed in the rrm3∆ mutant, albeit to a lesser extent than Top2, Rdh54 and Rad5, and further supports the increased requirement for tight regulation of D-loop formation in the absence of Rrm3 [19,175].…”