Regulation of Structure-Specific Endonucleases in Replication Stress

Abstract

Replication stress results in various forms of aberrant replication intermediates that need to be resolved for faithful chromosome segregation. Structure-specific endonucleases (SSEs) recognize DNA secondary structures rather than primary sequences and play key roles during DNA repair and replication stress. Holliday junction resolvase MUS81 (methyl methane sulfonate (MMS), and UV-sensitive protein 81) and XPF (xeroderma pigmentosum group F-complementing protein) are a subset of SSEs that resolve aberrant replication structures. To ensure genome stability and prevent unnecessary DNA breakage, these SSEs are tightly regulated by the cell cycle and replication checkpoints. We discuss the regulatory network that control activities of MUS81 and XPF and briefly mention other SSEs involved in the resolution of replication intermediates.

Keywords: Mus81; XPF; replication stress; structure-specific endonuclease.

Figure 1

Structure-specific endonucleases (SSEs) in different phases of the cell cycle. Mus81 (methyl methane sulfonate (MMS) and UV-sensitive protein 81) activity in Saccharomyces cerevisiae and human cells is stimulated during G2-M transition (reviewed in [8]). In Schizosaccharomyces pombe, Mus81 is activated by DNA damage. Xeroderma pigmentosum group F complementing protein (XPF)-excision repair cross-complementing group 1 (ERCC1) (orthologs Rad1-Rad10^S.c. and Rad16-Swi10^S.p.) is important for various DNA repair pathways and cleaves replication intermediates during S and G2 phases [10]. Scaffold protein SLX4 with associating partner SLX1 interacts with MUS81-EME1 (essential meiotic endonuclease 1) and XPF-ERCC1 in human cells (reviewed in [11]) [12,13,14,15] and their orthologs in in S. cerevisiae (reviewed in [16]) [17,18,19,20]. In contrast, Slx4 does not affect Rad16-Swi10 in S. pombe [21]. Activity of Yen1^S.c. is prevented until anaphase by restricting its nuclear entry due to phosphorylation of nuclear localization signal (NLS) [22,23,24]. Due to nuclear export signal (NES), GEN1 in human cells is able to access chromosomes only after nuclear membrane breakdown during mitosis [25]. S. pombe do not have Yen1 ortholog (reviewed in [26]). FEN1 (flap endonuclease 1) (orthologs Rad27^S.c. and Rad2^S.p.) and FAN1 (Fanconi-associated nuclease I) (missing in S. cerevisiae) contribute to processing replication intermediates but cell cycle-dependent regulation of these SSEs are not well characterized (reviewed in [8]). Mms4 (methyl methane sulfonate sensitivity protein 4).

Figure 2

Mus81 regulation by cell cycle kinases. In S. cerevisiae, Mus81-Mms4 is phosphorylated by Cdc28^S.c. (CDK1 ortholog) and Cdc5^S.c. (PLK ortholog) kinases at the G2/M transition [48,49,50]. Scaffold protein Rtt107^S.c. (PTIP ortholog) associates with Dpb11^S.c. (TOPBP1 ortholog) and interacts with DDK which also phosphorylates Mms4^S.c. [52]. Rtt107-Dpb11-Slx4^S.c. complex associates with Mus81-Mms4^S.c. behind replication forks. In S. pombe, Mus81-Eme1^S.p. is phosphorylated by Cdc2^S.p. (CDK1 ortholog) which primes Eme1^S.p. for phosphorylation by Rad3^S.p. (ATR ortholog) upon DNA damage [53]. Mus81-Eme1^S.p. may be contributing to Chk1 activation in fission yeast as Mus81-deleted cells with replication defect are able to bypass Chk1 checkpoint [54]. In human cells, MUS81-EME1 activity peak during M phase after EME1 is phosphorylated by CDK1, PLK1 [12,58,59]. SLX4 phosphorylation by CDK1 and MUS81 phosphorylation by CK2 also promotes MUS81-EME1 activity [60]. During S-phase, WEE1 downregulates MUS81-EME1 activity by inhibiting CDK1 and thereby limiting EME1 and SLX4 phosphorylation (reviewed in [56]). WEE1 inhibition of CDK2 reduces origin firing and subsequently the replication intermediate substrates of MUS81. WEE1 may also inhibit MUS81 directly [61]. Residual MUS81 activity during S-phase comes from MUS81 that forms complex with EME2 which can promote premature entry to mitosis upon WEE1 inhibition [62].

Figure 3

Mus81 regulation by replication checkpoint. In yeast, replication stress induces Rad3^S.p./Mec1^S.c. (ATR in human) activation of Cds1^S.p./Rad53^S.c. by promoting its association with Mrc1^S.p./S.c. (CLASPIN in human) (reviewed in [80]). Upon acute and severe replication stress such as hydroxyurea treatment, Cds1^S.p. limits Mus81^S.p. activity (indicated by solid red line) [44]. Cds1^S.p. inhibits Rad60^S.p. activity (indicated by blue line) by promoting delocalization from the nucleus [82,83]. Mrc1^S.p./S.c. protein level regulates recruitment of Rqh1^S.p. homolog Sgs1^S.c. to chromatin (indicated by blue arrow) [74]. Both Rad60^S.p./Esc2^S.c. and Rqh1^S.p./Sgs1^S.c. contribute to Mus81 activity (indicated by dashed blue arrow) [75,76]. In human cells, DNA damage checkpoint CHK1 and Cds1-homolog CHK2 is activated downstream of ATM/ATR kinases (reviewed in [79,80]) [77]. It is unclear whether MUS81 is directly regulated by these checkpoint kinases in human cells. However, there is evidence that CHK2 upregulates MUS81 protein levels and MUS81 in turn contributes to CHK2 activation upon DNA damage (indicated by dashed double-headed arrow) [84]. Deleterious MUS81-dependent processing of replication intermediates following CHK1 inhibition suggests that CHK1 downregulates MUS81 activity (indicated by dashed red line) [68,69,70].

Figure 4

Important domains in SLX4. Across all three species (S.p., S.c., and human), scaffold protein SLX4 have SLX1 binding domain (SBD) and MUS81-EME1 binding region (SAP) [58]. Slx4^S.c. in budding yeast and SLX4 in human cells also have XPF-ERCC1 interacting region MLR. Slx4^S.p. in fission yeast lack MLR. SLX4 in human cells have UBZ (ubiquitin-binding zinc finger domain) and SIM (SUMO-interaction motif )motifs that contributes to its recruitment and activity [135,136]. (SBD: SLX1 binding domain; SAP: SAF-A/B, Acinus and PIAS domain that interacts with MUS81-EME1; MLR: MEI9XPF-interaction-like region that interacts with XPF-ERCC1; ICL: interstrand crosslink; CFS: common fragile site) (S.c.: S. cerevisiae; S.p.: S. pombe).

Figure 5

Summary of types of regulation of SSEs (MUS81 and XPF in particular) involved in resolving replication intermediates.