Structure-Specific Endonucleases and the Resolution of Chromosome Underreplication

Abstract

Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.

Keywords: DNA replication; Holliday junction resolvase; chromosome segregation; chromosome stability; genome stability; mitotic DNA synthesis; replication stress; structure-specific nuclease; ultrafine anaphase bridge.

Figure 1

Human structure-specific endonuclease (SSE) domain structures and DNA substrate specificities. MUS81 and its alternative binding partners EME1 and EME2 (length in amino acids is indicated) contain ERCC4 endonuclease domain and helix-hairpin-helix (H) motifs (gray font denotes degenerate motifs). While MUS81-EME1 exhibits activity on 3′-flaps, replication forks (RFs) and nicked Holliday junctions (HJs) (red arrows), MUS81-EME2 additionally cleaves D-loop strand-invasion structures and 5′-flaps as well as being more active on intact HJs. As part of a SLX-MUS complex (see text), MUS81-EME1 effectively resolves HJs by symmetric cleavage after pre-nicking mediated by the SLX1-SLX4 nuclease. GEN1 contains N-terminal and internal XPG nuclease motifs (X_N and X_I), followed by a 5′-3′ exonuclease domain (EXO) and a chromodomain (CD) that promotes substrate recognition [71]; N denotes a nuclear export signal. GEN1 cuts 5′-flaps, RFs and HJs. SLX1 is a GIY-YIG nuclease with a zinc-finger (ZF) at the C-terminus. Associated with SLX4 via a C-terminal SLX1-binding domain (SBD), SLX1 cleaves splayed arm, 5′-flap, RF and HJ substrates. SLX4 contains a ZF domain (two copies of ubiquitin-binding UBZ4), multiple SUMO-interacting motifs (SIMs) [72] and scaffolds a tri-nuclease complex that is known as SMX containing SLX1, MUS81-EME1 (bound at its SAP domain) and ERCC1-XPF (bound via MLR, BTB). Figure is modified from [5].

Figure 2

Multiple roles of SSEs in RF recovery. SSEs target replication (RI) and HR (HRI) intermediates to facilitate replication restart and completion. At DNA lesions, RF arrest may be overcome by HR-mediated bypass. Re-initiation of DNA synthesis downstream of lesions leaves daughter-strand gaps that are subsequently filled in by template switching. The ensuing HRIs may be removed by Sgs1/BLM-dependent dissolution (not depicted on figure) or cleavage by SSEs. RF reversal by disengagement of the leading and lagging strands at stalled forks followed by nascent-strand annealing generates HJ-like DNA four-way RIs. These intermediates are shielded from degradation, which facilitates passive rescue by converging RFs. If reversed RFs are not permanently inactivated, such as by replisome loss, remodeling for direct restart that is mediated by DNA helicases/translocases may be possible. Alternatively, functional RFs are restored by HR-mediated restart through invasion of the upstream template and associated HRIs are removed by SSEs. Persistent RIs have emerged as important non-HRI targets of SSEs. The cleavage of RF structures produces single-ended DNA double-strand breaks, triggering break-induced replication (BIR). Invasion of the unbroken sister chromatid generates a D-loop and subsequently a new processive RF. HRIs formed along the BIR pathway are once again resolved by SSEs.

Figure 3

SSEs promote sister chromatid disjunction and replication completion. RS leads to an accumulation of unresolved replication (RI) and HR (HRI) intermediates linking nascent sister chromatids. If replication is not completed in S phase and not all RIs and HRIs are removed, SSEs resolve persistent intermediates in mitosis. RI cleavage initiates late DNA repair synthesis along the mitotic DNA synthesis (MiDAS) pathways, which promotes replication completion in mitotic cells and safeguards sister chromatid disjunction. Failure to resolve RI and HRI sister chromatid DNA links leads to BLM and PICH-bound UFBs, mitotic DNA damage and segregation failure.