Multifunctional roles of Saccharomyces cerevisiae Srs2 protein in replication, recombination and repair

Abstract

The Saccharomyces cerevisiae Srs2 DNA helicase has important roles in DNA replication, recombination and repair. In replication, Srs2 aids in repair of gaps by repair synthesis by preventing gaps from being used to initiate recombination. This is considered to be an anti-recombination role. In recombination, Srs2 plays both prorecombination and anti-recombination roles to promote the synthesis-dependent strand annealing recombination pathway and to inhibit gaps from initiating homologous recombination. In repair, the Srs2 helicase actively promotes gap repair through an interaction with the Exo1 nuclease to enlarge a gap for repair and to prevent Rad51 protein from accumulating on single-stranded DNA. Finally, Srs2 helicase can unwind hairpin-forming repeat sequences to promote replication and prevent repeat instability. The Srs2 activities can be controlled by phosphorylation, SUMO modification and interaction with key partners at DNA damage or lesions sites, which include PCNA and Rad51. These interactions can also limit DNA polymerase function during recombinational repair independent of the Srs2 translocase or helicase activity, further highlighting the importance of the Srs2 protein in regulating recombination. Here we review the myriad roles of Srs2 that have been documented in genome maintenance and distinguish between the translocase, helicase and additional functions of the Srs2 protein.

Keywords: DNA helicase; DNA repair, recombination; Srs2, toxic recombination; anti-recombinase; translocase.

Figure 1.

Role of Srs2 in the promotion of PRR. PCNA interacts with DNA polymerases to facilitate processive leading and lagging strand DNA synthesis. DNA replication can stall when a DNA lesion is encountered, and exposure of excess single-stranded DNA may lead to Rad51-dependent salvage recombination events. Sumoylation of PCNA at K164 and K127 by Ubc9 and Siz1 recruits Srs2 helicase, which actively dismantles Rad51 filament on the single-stranded DNA and antagonizes HR. Replication fork stalling also triggers monoubiquitylation of PCNA in a manner that depends on Rad6 and Rad18. Monoubiquitylated PCNA recruits translesion DNA polymerases to bypass the DNA lesion through translesion synthesis pathway (TLS). The Ubc13-Mms2-Rad5 E2 and E3 ensemble can further polyubiquitylate PCNA, which activates template switching and error-free lesion bypass.

Figure 2.

Scheme of Srs2 domain organization. (A) The UvrD like helicase domain is marked in green. The motifs mediating interaction with Rad51, PCNA and SUMO are marked in purple, pink and yellow, respectively. K41 is a key residue in the Walker A motif and is required for ATP binding. Mutation of K41 to alanine inactivates the helicase and translocase activities of Srs2. The seven Cdk1 phosphorylation sites are labeled with a star, and the locations of three lysine residues that are sumoylated in vivo were marked with solid square. (B) A comparison of Srs2 homologs from multiple fungal species shows high conservation in the N-terminal helicase domain, but greater variation at the C-terminus. Helicase domains are colored in green. PCNA and SUMO-binding motifs are colored in pink and yellow, respectively. Notably, Kluyveromyces lactis contains two Srs2 homologs with C-terminus in difference length. (S. cer: Saccharomyces cerevisiae; Z. rou: Zygosaccharomyces rouxii; C. gla: Candida glabrata; A. gos: Eremothecium gossypii; K. lac: Kluyveromyces lactis; D. han: Debaryomyces hansenii; Y. lip: Yarrowia lipolytica; S. pomb: Schizosaccharomyces pombe.

Figure 3.

Anti-recombination and anti-crossover activities of Srs2 during replication and HR mediated DSB repair. (A) Accumulation of single-stranded DNA at stalled replication forks can lead to Rad51 filament assembling and Rad51-catalyzed recombination events. PCNA, when sumoylated, actively recruits Srs2 helicase to turn over Rad51 filaments and suppress salvage recombination events. (B) Double-strand breaks (DSBs) can be repaired by HR, which is initiated by the generation of 3΄ single-stranded DNA through DNA end resection. RPA, the single-stranded DNA-binding protein loaded during DNA end resection, is replaced by Rad51, a reaction facilitated by mediator activities from Rad52 and Rad55-Rad57. Rad51 is able to catalyzed strand invasion when Rad54 is present. In its absence, the Rad51 filament may become toxic and requires strippase activity from Srs2 for its turnover, so that alternative repair mechanisms can be employed. Upon successful strand invasion, the invaded 3΄ end is extended by repair DNA synthesis to recover the missing genetic information based on the available homologous template. The resulting intermediates may be processed through SDSA pathway where the invading strand is displaced and annealed to the opposite 3΄ end of the break followed by gap filling. Multiple lines of genetic evidence suggest Srs2 plays a key role in SDSA pathway.

Figure 4.

Srs2 catalyzed triplet-repeat hairpin unwinding during replication. Triplet-repeat hairpins form when single-stranded DNA is exposed during replication and may cause expansion or contraction of the repetitive sequences. Srs2 is recruited by PCNA to actively unfold the triplet-repeat hairpin so as to maintain the stability of the repetitive sequences.

Figure 5.

Pathways for removing misincorporated ribonucleotides during replication. Ribonucleotides are incorporated into the genome by replicative DNA polymerases, primarily on the leading strand. A majority of the embedded rNMPs are removed by ribonucleotide excision repair (RER), a coupled reaction of RNase H2, DNA polymerase, Fen1/Exo1 endonuclease and DNA ligase I (Cdc9) as depicted (A). The misincorporated ribonucleotides that escaped from RER pathway are cleaved by Top1 to create nicks with a 3΄ end that is terminated by a 2΄, 3΄-cyclic phosphate and a 5΄-end terminated by a 5΄-hydroxyl group (B). The 2΄, 3΄-cyclic phosphate terminated nicks, although unligatible, may be processed by Top1 again and cause formation of slippage mutations when the misincorporation occurs in short repetitive DNA regions (C). Srs2 helicase and Exo1 nuclease together are able to generate a gap at the 5΄ end of the nick, which prevents Top1-mediated error-prone repair and facilitates error-free gap repair (D).