Structure-selective Endonucleases Research Articles

Coordinated Actions of SLX1-SLX4 and MUS81-EME1 for Holliday Junction Resolution in Human Cells

Holliday junctions (HJs) are four-way DNA intermediates that form during homologous recombination, and their efficient resolution is essential for chromosome segregation. Here, we show that three structure-selective endonucleases, namely SLX1-SLX4, MUS81-EME1, and GEN1, define two pathways of HJ resolution in human cells. One pathway is mediated by GEN1, whereas SLX1-SLX4 and MUS81-EME1 provide a second and genetically distinct pathway (SLX-MUS). Cells depleted for SLX-MUS or GEN1 pathway proteins exhibit severe defects in chromosome segregation and reduced survival. In response to CDK-mediated phosphorylation, SLX1-SLX4 and MUS81-EME1 associate at the G2/M transition to form a stable SLX-MUS holoenzyme, which can be reconstituted invitro. Biochemical studies show that SLX-MUS is a HJ resolvase that coordinates the active sites of two distinct endonucleases during HJ resolution. This cleavage reaction is more efficient and orchestrated than that mediated by SLX1-SLX4 alone, which exhibits a potent nickase activity that acts promiscuously upon DNA secondary structures.

An elegans Solution for Crossover Formation

An elegans Solution for Crossover Formation

Complex Chromosomal Rearrangements Mediated by Break-Induced Replication Involve Structure-Selective Endonucleases

DNA double-strand break (DSB) repair occurring in repeated DNA sequences often leads to the generation of chromosomal rearrangements. Homologous recombination normally ensures a faithful repair of DSBs through a mechanism that transfers the genetic information of an intact donor template to the broken molecule. When only one DSB end shares homology to the donor template, conventional gene conversion fails to occur and repair can be channeled to a recombination-dependent replication pathway termed break-induced replication (BIR), which is prone to produce chromosome non-reciprocal translocations (NRTs), a classical feature of numerous human cancers. Using a newly designed substrate for the analysis of DSB–induced chromosomal translocations, we show that Mus81 and Yen1 structure-selective endonucleases (SSEs) promote BIR, thus causing NRTs. We propose that Mus81 and Yen1 are recruited at the strand invasion intermediate to allow the establishment of a replication fork, which is required to complete BIR. Replication template switching during BIR, a feature of this pathway, engenders complex chromosomal rearrangements when using repeated DNA sequences dispersed over the genome. We demonstrate here that Mus81 and Yen1, together with Slx4, also promote template switching during BIR. Altogether, our study provides evidence for a role of SSEs at multiple steps during BIR, thus participating in the destabilization of the genome by generating complex chromosomal rearrangements.

Distinct Roles of Mus81, Yen1, Slx1-Slx4, and Rad1 Nucleases in the Repair of Replication-Born Double-Strand Breaks by Sister Chromatid Exchange

Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.

Three Structure-Selective Endonucleases Are Essential in the Absence of BLM Helicase in Drosophila

DNA repair mechanisms in mitotically proliferating cells avoid generating crossovers, which can contribute to genome instability. Most models for the production of crossovers involve an intermediate with one or more four-stranded Holliday junctions (HJs), which are resolved into duplex molecules through cleavage by specialized endonucleases. In vitro studies have implicated three nuclear enzymes in HJ resolution: MUS81–EME1/Mms4, GEN1/Yen1, and SLX4–SLX1. The Bloom syndrome helicase, BLM, plays key roles in preventing mitotic crossover, either by blocking the formation of HJ intermediates or by removing HJs without cleavage. Saccharomyces cerevisiae mutants that lack Sgs1 (the BLM ortholog) and either Mus81–Mms4 or Slx4–Slx1 are inviable, but mutants that lack Sgs1 and Yen1 are viable. The current view is that Yen1 serves primarily as a backup to Mus81–Mms4. Previous studies with Drosophila melanogaster showed that, as in yeast, loss of both DmBLM and MUS81 or MUS312 (the ortholog of SLX4) is lethal. We have now recovered and analyzed mutations in Drosophila Gen. As in yeast, there is some redundancy between Gen and mus81; however, in contrast to the case in yeast, GEN plays a more predominant role in responding to DNA damage than MUS81–MMS4. Furthermore, loss of DmBLM and GEN leads to lethality early in development. We present a comparison of phenotypes occurring in double mutants that lack DmBLM and either MUS81, GEN, or MUS312, including chromosome instability and deficiencies in cell proliferation. Our studies of synthetic lethality provide insights into the multiple functions of DmBLM and how various endonucleases may function when DmBLM is absent.

Meiotic Recombination Intermediates Are Resolved with Minimal Crossover Formation during Return-to-Growth, an Analogue of the Mitotic Cell Cycle

Accurate segregation of homologous chromosomes of different parental origin (homologs) during the first division of meiosis (meiosis I) requires inter-homolog crossovers (COs). These are produced at the end of meiosis I prophase, when recombination intermediates that contain Holliday junctions (joint molecules, JMs) are resolved, predominantly as COs. JM resolution during the mitotic cell cycle is less well understood, mainly due to low levels of inter-homolog JMs. To compare JM resolution during meiosis and the mitotic cell cycle, we used a unique feature of Saccharomyces cerevisiae, return to growth (RTG), where cells undergoing meiosis can be returned to the mitotic cell cycle by a nutritional shift. By performing RTG with ndt80 mutants, which arrest in meiosis I prophase with high levels of interhomolog JMs, we could readily monitor JM resolution during the first cell division of RTG genetically and, for the first time, at the molecular level. In contrast to meiosis, where most JMs resolve as COs, most JMs were resolved during the first 1.5–2 hr after RTG without producing COs. Subsequent resolution of the remaining JMs produced COs, and this CO production required the Mus81/Mms4 structure-selective endonuclease. RTG in sgs1-ΔC795 mutants, which lack the helicase and Holliday junction-binding domains of this BLM homolog, led to a substantial delay in JM resolution; and subsequent JM resolution produced both COs and NCOs. Based on these findings, we suggest that most JMs are resolved during the mitotic cell cycle by dissolution, an Sgs1 helicase-dependent process that produces only NCOs. JMs that escape dissolution are mostly resolved by Mus81/Mms4-dependent cleavage that produces both COs and NCOs in a relatively unbiased manner. Thus, in contrast to meiosis, where JM resolution is heavily biased towards COs, JM resolution during RTG minimizes CO formation, thus maintaining genome integrity and minimizing loss of heterozygosity.

Erratum to: Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Erratum to: Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes.

Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81–Mms4/EME1, Slx1–Slx4/BTBD12/MUS312, XPF–ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.

Three Structure-Selective Endonucleases Are Essential in the Absence of BLM Helicase in Drosophila

Three Structure-Selective Endonucleases Are Essential in the Absence of BLM Helicase in Drosophila

A junction branch point adjacent to a DNA backbone nick directs substrate cleavage by Saccharomyces cerevisiae Mus81-Mms4

The DNA structure-selective endonuclease Mus81-Mms4/Eme1 incises a number of nicked joint molecule substrates in vitro. 3′-flaps are an excellent in vitro substrate for Mus81-Mms4/Eme1. Mutants in MUS81 are synthetically lethal with mutations in the 5′-flap endonuclease FEN1/Rad27 in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Considering the possibility for isoenergetic interconversion between 3′- and 5′- flaps, these data are consistent with the hypothesis that Mus81-Mms4/Eme1 acts on 3′-flaps in vivo. FEN1/Rad27 prefers dually flapped substrates and cleaves in a way that allows direct ligation of the resulting nick in the product duplex. Here we test the activity of Mus81-Mms4 on dually flapped substrates and find that in contrast to FEN1/Rad27, Mus81-Mms4 activity is impaired on such substrates, resulting in cleavage products that do not allow direct religation. We conclude that Mus81-Mms4, unlike FEN1/Rad27, does not prefer dually flapped substrates and is unlikely to function as a 3′-flapase counterpart to the 5′-flapase activity of FEN1/Rad27. We further find that joint molecule incision by Mus81-Mms4 occurs in a fashion determined by the branch point, regardless of the position of an upstream duplex end. These findings underscore the significance of a nick adjacent to a branch point for Mus81-Mms4 incision.

Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease.

The DNA structure-selective endonuclease Mus81-Mms4/Eme1 is a context-specific recombination factor that supports DNA replication, but is not essential for DSB repair in Saccharomyces cerevisiae. We overexpressed Mus81-Mms4 in S. cerevisiae, purified the heterodimer to apparent homogeneity, and performed a classical enzymological characterization. Kinetic analysis (kcat, KM) demonstrated that Mus81-Mms4 is catalytically active and identified three substrate classes in vitro. Class I substrates reflect low KM (3–7 nM) and high kcat (∼1 min−1) and include the nicked Holliday junction, 3′-flapped and replication fork-like structures. Class II substrates share low KM (1–6 nM) but low kcat (≤0.3 min−1) relative to Class I substrates and include the D-loop and partial Holliday junction. The splayed Y junction defines a class III substrate having high KM (∼30 nM) and low kcat (0.26 min−1). Holliday junctions assembled from oligonucleotides with or without a branch migratable core were negligibly cut in vitro. We found that Mus81 and Mms4 are phosphorylated constitutively and in the presence of the genotoxin MMS. The endogenous complex purified in either modification state is negligibly active on Holliday junctions. Hence, Holliday junction incision activity in vitro cannot be attributed to the Mus81-Mms4 heterodimer in isolation.

Biochemical analysis of Saccharomyces cerevisiae Mus81‐Mms4, an endonuclease that supports eukaryotic DNA replication

Saccharomyces cerevisiae Mus81-Mms4 is a DNA structure-selective endonuclease that functions in a number of contexts that use the DNA homology search and strand exchange properties of the RAD52 epistasis group. Unique to eukaryotes, the Mus81 endonuclease contributes to tolerance of agents that impair replication fork progression but, unusual for its association with the RAD52 recombination pathway, the complex does not appear to contribute to DNA double-strand break repair outside of meiosis. A number of branched DNA structures can be entertained as potential in vivo substrates of Mus81-Mms4, but whether the heterodimer acts on Holliday junctions, 3′-flaps, displacement (D-) loops, nicked junctions, or other recombination intermediates in vivo cannot be extrapolated with confidence from its biochemical behavior. We overexpressed Mus81-Mms4 in S. cerevisiae and purified the heterodimer to apparent homogeneity, and performed Michaelis-Menten rate studies to determine the catalytic parameters Km and kcat on a number of cleavable substrates. The Km values, although low, exceed the expected in vivo substrate concentration and may indicate that an additional factor(s) may target the complex to substrates in vivo. We are currently testing whether the ssDNA binding protein RPA and the recombination proteins Rad51 and Rad54, which may be present on joint molecules they generate in vivo, contribute to the catalytic properties of Mus81-Mms4. Funded by NIH-RO1