Resolving branched DNA intermediates with structure-specific nucleases during replication in eukaryotes

Abstract

Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81-Mms4/MUS81-EME1, Yen1/GEN1, and Slx1-Slx4/SLX1-SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication process.

Fig. 1

DNA replication problems can lead to replication fork stalling and unreplicated chromosomal areas (i), and to the formation of branched repair intermediates from homologous recombination-dependent RF recovery pathways (ii). The resulting physical links between sister chromatids can be targeted by structure-specific nucleases to facilitate the completion of S phase and chromosome segregation (iii)

Fig. 2

DNA double-strand break repair and replication fork support mediated by homologous recombination. Steps 1–6 describe the canonical DSB repair model of HR. 1, DNA end resection produces 3′-single-stranded overhangs which are bound by strand exchange protein Rad51/RAD51; 2, the resulting nucleoprotein-filament is capable of identifying and invading a homologous donor duplex, thereby creating a displacement-loop (D-loop) and initiating repair synthesis; 3, second-end capture, the association of the D-loop with the non-invading break end, also initiates repair synthesis; 4, after repair and nick ligation, the recombining molecules are joined together at a double HJ intermediate; 5, HJ resolvases introduce symmetrically related nicks (blue arrowheads) to produce discrete duplex products; 6, resolution of a pair of HJs along the same axis produces non-crossover products, while the use of different axes leads to crossover products characterized by reciprocal genetic exchange of flanking markers (as shown). Double HJ dissolution offers an alternative to nucleolytic cleavage: 7, the Sgs1–Top3–Rmi1/BLM–TOPOIIIα–RMI1–RMI2 complex drives HJ convergence by branch migration and removal of the resulting hemi-catenate; 8, dissolution results exclusively in non-crossover products, which differ from the DNA molecules at outset only by the presence of a repair patch. 9, Synthesis-dependent strand annealing (SDSA). Disassembly of the D-loop by expulsion of the invading DNA single-strand frees up a repair template for the second break end. 10–16, HR facilitates DNA replication: 10, a range of replication blocks may stall RF progression (see text); 11, a blocked RF (gray) can regress to form a HJ-like structure with a recombinogenic DSB end homologous to the DNA ahead of the four-way DNA junction; 12, HR-mediated strand-invasion forms a D-loop at which DNA synthesis may be re-initiated. HJs that arise during the process can be removed by nucleolytic cleavage (blue arrowheads) to reestablish a processive RF (13). 14, A lesion in the lagging strand template (pink sphere) can be bypassed without RF arrest; 15, the lesion is tolerated at the cost of a single-stranded DNA gap known as daughter strand gap; 16, HR-mediated gap repair and HJ formation. HJ resolution (blue arrowheads) or dissolution can restore a normal RF (13)

Fig. 3

Replication fork recovery by simple resetting or deliberate breakage. 1, RF movement can be impeded by a range of factors (see text); 2, potentially leading to RF arrest (inactive fork, gray); 3, RF regression, by annealing of the nascent leading and lagging strands with one another, leads to the formation of a HJ-like structure. Template switching allows leading strand extension (indicated by the triple arrowhead) if the lagging strand had been extended further than the leading stand prior to regression. This might occur via the RAD6–RAD18–RAD5 DNA damage tolerance pathway when a RF is blocked at a lesion in the leading strand template (Ghosal and Chen 2013). Regression also ensures that any blocking lesion is moved away from the fork and placed back into a duplex DNA context enabling excision repair pathways. 4, If the cause for regression can be removed (or if template switch enables bypass/DNA damage tolerance), HJ branch migration may reset an active fork (blue). Alternatively, deliberate fork cleavage might occur: 5, the substrate spectra of eukaryotic structure-specific nucleases such as Mus81–Mms4/MUS81–EME1 may allow them to act directly on RFs (red arrowhead); 6, RF cleavage may also occur after conversion of the three-way junction into a four-way HJ intermediate. 7–10, Recombination-dependent replication/break-induced replication (BIR) pathways can reestablish a processive RF from a single-ended DSB after RF breakage (or spontaneous collapse at a preexisting nick in the template). Note that this pathway entails the formation of a single HJ (step 9), which requires the attention of a structure-specific nuclease (blue arrowheads)

Fig. 4

Domain structure and in vitro DNA substrate specificities of the eukaryotic HJ-resolving enzymes. These structure-specific nucleases are largely conserved from yeast to human (Schwartz and Heyer 2011), with the human proteins depicted (number of amino acid residues in brackets). MUS81 and EME1 contain an excision repair cross complementation group 4 (ERCC4) endonuclease domain and helix-hairpin-helix (H) motifs (gray font denotes degenerate/inactive motifs). The MUS81–EME1 heterodimer exhibits activity towards 3′-flaps, RFs, and HJ substrates (red arrowheads). HJs are efficiently resolved if they contain a preexisting nick. GEN1 is a monomeric XPG family nuclease that contains the superfamily-specific N-terminal and internal XPG nuclease motifs (X_N and X_I, respectively). Adjacent to the nuclease domain is a helix-hairpin-helix motif. GEN1 cuts 5′-flaps, RFs, and HJs, as indicated. SLX1 is a small GIY-YIG superfamily nuclease with a PHD-type zinc-finger (ZF) motif at the C-terminus. It forms a heterodimeric structure-specific nuclease with the large SLX4 subunit. SLX4 contains ubiquitin-binding zinc-finger-, MUS312-MEI9 interaction-like (MLR)-, bric-a-brac tramtrack broad complex (BTB)-, SAF-A/B acinus and PIAS (SAP)-, and SLX1-binding (SBD) domains. Human SLX4 interacts with multiple nucleases involved in ICL repair via the Fanconi anemia pathway, as indicated below. The interaction with SNM1B (Salewsky et al. 2012) has not been mapped. SLX1–SLX4 cleaves splayed arm, 5′-flap, RF, and HJ substrates. HJs are cleaved in symmetric fashion by the human protein, but nicked at multiple nonsymmetric positions by yeast Slx1–Slx4 (orange arrowheads)

Fig. 5

Ways in which the HJ-resolving endonucleases Mus81–Mms4/MUS81–EME1, Yen1/GEN1, and Slx1–Slx4/SLX1–SLX4 may support DNA replication. When cells progress through S phase, RF stalling and arrest can lead to the accumulation of replication intermediates (RIs) not actively engaged in DNA synthesis. 1, MUS81-dependent DSB formation in response to drug-induced (APH, CPT, HU, MMC) or oncogene-induced (overexpressed cyclin D1, E) replication stress indicates active RI cleavage. Subsequent replication restart and increased cell survival has been reported, suggesting that active RF breakage can promote bulk DNA synthesis, while unrestrained RI cleavage may cause cell death. CDK1-dependent stimulation of MUS81 activity may promote DSB-independent RF recovery in early S phase and delay RF breakage until replication is approaching its completion. Reversed RFs with strand interruptions have been observed in unperturbed human cells and may represent an in vivo target for MUS81–EME1 in accordance with the enzyme's in vitro preference for nicked HJs. Given the functional overlap between Mus81 and Yen1 in yeast, it is conceivable that GEN1 also targets RIs. 2, RFs stalled within difficult-to-replicate areas, such as common fragile sites and the rDNA array in yeast, have been shown to require the attention of HJ-resolving enzymes. Fragile site cleavage by MUS81 (red arrowhead) allows the disengagement of the parental DNA strands within unreplicated segments and sister chromatid disjunction. A higher incidence of anaphase bridges in cells depleted for GEN1 indicates the enzyme may fulfill related functions (cutting RFs with the opposite polarity, orange arrowhead). Slx1–Slx4 has been proposed to resolve blocked RFs in the rDNA in yeast (purple arrowheads). A similar reaction may be catalyzed by SLX4 in conjunction with multiple nucleases at RFs arrested at intrastrand crosslinks (FA pathway). 3, HR intermediates (HRIs) arise during RF recovery and DSB repair (see Figs. 2 and 3, steps 7–10). In yeast, Mus81–Mms4 and Yen1 have been shown to resolve HRIs upon replication stress and under unperturbed conditions. Perhaps the most likely scenario is that Mus81–Mms4 targets nicked HJ precursors and D-loop structures by virtue of its 3′-flap cleavage activity. After maturation into fully ligated HJs, Yen1 is the more suitable processing factor (shown here on a double HJ intermediate). This is in agreement with a successive cell cycle-dependent surge in Mus81–Mms4 and Yen1 activity as cells approach G2/M (below, maximal activity bright red)