Cohesin ATPase activities regulate DNA binding and coiled-coil configuration

The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs.

The cohesin complex is required for sister chromatid cohesion, DNA damage response, gene expression, and spatial organization of the genome (1, 2). Psm1/SMC1 and Psm3/SMC3 form a stable heterodimer via both hinge–hinge interaction and ATPase heads engagement upon ATP binding (3–5). Cohesin owns two ATPase domains at its globular head. Each ATPase domain contains the Walker A and Walker B consensus sequences found in most ATPases (5, 6) and several other sequence motifs, such as signature motif and D loop (7). Both ATPase domains are required for efficient loading of cohesin (8). Rad21/SCC1, the kleisin subunit with its N-terminal domain, interacts with Psm3/SMC3 coiled coils (CCs) emerging from the head, and its C-terminal domain interacts with Psm1/SMC1 head domain (9–12). Psc3/SCC3 associates with the unstructured region in the middle of Rad21/SCC1 (13–15).Mis4/SCC2/NIPBL functions as the cohesin loader (16, 17). Mis4/SCC2/NIPBL forms a harp-shaped structure (18, 19). Its N-terminal domain binds to Psm3/SMC3 coiled coils close to the head domain and its C-terminal domain binds to Psm1/SMC1 coiled coils close to the head domain (11, 15). Mis4/SCC2/NIPBL also stimulates cohesin’s ATPase activity for efficient cohesin loading (20–22).All coiled coils of SMC complexes (cohesin, condensin, and SMC5-SMC6 complex) are ∼50 nm long and are essential for their functions (23–25). SMC coiled coils contain interruptions (break sites hereafter) that disrupt the characteristic seven-residue amino acid sequence repeats, known as heptad repeats (26, 27). It has been proposed that cohesin folds around the midpoints of its coiled coils to bring the head and hinge domains into proximity (20, 28–30). However, it is still unclear how such molecular architecture of cohesin works to fulfill its function. In this study, we isolated temperature-sensitive (ts) mutants with single amino acid substitutions in the signature motif or D loop of cohesin ATPase domains, which presumably impair ATPase activity of cohesin. Then, screening of suppressor mutations that rescued the lethality caused by ATPase defects identified several hot regions in cohesin SMC subunits, which are involved in DNA binding, interaction with non-SMC subunits, or coiled-coil dynamics around midpoints. Therefore, these results coupled the dynamics of the cohesin complex with ATPase activity.     

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.Replisomes are multisubunit protein complexes that coordinately unwind duplex DNA and duplicate both parental strands during chromosomal replication. Detailed studies of cellular and viral systems show that the basic functional units of replication—helicase, primase, and DNA polymerase (Pol)—are common to all replisomes whereas the evolutionary histories of the individual components in different kingdoms are distinctive and diverse (1). Accordingly, the sequence and structure of replisome components are unrelated, and thus connections and coordination among the different functional units can be expected to vary widely.The most well-studied cellular replisome to date, bacterial Escherichia coli, uses multiple copies of a single DNA polymerase to replicate both parental strands, and the action of these polymerases is coordinated by a multifunctional clamp loader that also connects to the replicative helicase (2). For reasons that are still unclear, the eukaryotic replisome uses three different polymerases for normal chromosome duplication, including one for the leading strand (Pol ε) and two for the lagging strand (Pol α/primase and Pol δ) (3–5). Similarly, whereas the replicative helicase in E. coli is a homohexamer of DnaB, the eukaryotic CMG (Cdc45-MCM-GINS) helicase consists of 11 distinct subunits assembled on chromatin by loading of the heterohexameric Mcm2-7 helicase core at an origin and its subsequent activation by association with Cdc45 and the heterotetrameric GINS (Sld5-Psf1-Psf2-Psf3) complex at the onset of S-phase to form the CMG complex (6–8). Among other things, the complexity of the eukaryotic system reflects the need to restrict chromosome duplication to a single round in a normal cell cycle so that proper ploidy can be maintained across multiple chromosomes after cell division.Detailed biochemical studies of the E. coli replisome show that the leading and lagging strand replicases are coupled and intimately linked to the replicative helicase, a feature also common to the well-characterized T4 and T7 bacteriophage replication systems (9–11). For this reason, it has been assumed that the same would be true of eukaryotic systems, and this notion has been strongly reinforced by the identification in yeast of replication progression complexes (RPCs), large multiprotein complexes containing, among other proteins, CMG, Mcm10, Mrc1, and Ctf4 (12, 13). The RPC also contains Pol α/primase under low-salt conditions, suggesting that it is more weakly bound, and binding of Pol α to the replisome is abolished in cells lacking Ctf4 or its metazoan counterpart, AND-1 (13–16). Ctf4 binds both the catalytic Pol1 subunit of Pol α and GINS in yeast and thus is thought to tether Pol α to CMG in the replisome (13, 16, 17).Neither Pol δ nor Pol ε is found in the most highly purified RPCs, which are defined by mass spectrometry of proteins bound after sequential affinity purification of two separate CMG components from a cell extract (12, 13). However, the noncatalytic Dpb2 protein subunit of Pol ε is known to bind to the GINS component of CMG, and recent evidence suggests that this interaction helps maintain Pol ε at the replication fork (18, 19). Pol δ was shown to bind Pol α via its nonessential Pol32 subunit (20), suggesting that Pol δ might be recruited from solution to extend primers initiated by Pol α/primase and may only associate transiently with the core replisome.To study the eukaryotic replisome in detail, we initiated a long-term project to purify the numerous components of the RPC/replisome from the model eukaryote Saccharomyces cerevisiae. Pioneering work on Drosophila and human CMG showed that an active helicase complex could be obtained by coexpression of all 11 subunits in insect cells (7, 21) so we cooverexpressed all 11 CMG subunits in yeast and purified the complex to homogeneity (22). We showed that, like its human counterpart, yeast CMG is capable of catalyzing replication of a model replication-fork substrate (21, 22). Using this system, we also showed that CMG enforces a preference for Pol ε over Pol δ in leading-strand replication whereas proliferating cell nuclear antigen (PCNA) enforces the opposite preference on the lagging strand (22). Preferential binding of Pol δ to PCNA has been clearly demonstrated and provides an explanation for the dominance of Pol δ in lagging-strand synthesis (23), but the nature of any interaction between Pol ε and CMG on the leading strand is poorly understood.While purifying CMG from yeast, we identified a direct interaction between overexpressed CMG and native Pol ε to form a multifunctional eukaryotic leading-strand holoenzyme that we refer to as CMGE. Using separately purified CMG and Pol ε, we reconstituted a stable, 15-subunit CMGE and showed that it is an active helicase–polymerase in vitro. We also show that the Dpb2 subunit of Pol ε, which binds to the Psf1 protein subunit of GINS, promotes efficient Pol ε function with CMG. Direct binding of Pol ε to the full CMG complex has not been previously demonstrated, and this interaction provides a mechanistic foundation for preferential replication of the leading strand by Pol ε as part of a stable helicase–polymerase holoenzyme (4).     

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis

Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli β clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and β limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.Despite the action of several DNA repair pathways, unrepaired damage is encountered by replicative DNA polymerases, which stall at DNA-distorting lesions. Translesion synthesis (TLS), most notably by Y-family polymerases, is one pathway that alleviates such roadblocks. In TLS, a Y-family polymerase switches with a stalled replicative polymerase, synthesizes across from and past the lesion, and then, switches back to allow resumption of normal synthesis (1). The ability of Y-family polymerases to bypass damaged DNA comes at the cost of lower fidelity, requiring careful regulation of polymerase exchange (2).Processive synthesis by DNA polymerases requires their tethering to the protein-binding cleft of a ring-shaped processivity clamp by a conserved clamp-binding motif (CBM). Canonical clamps, such as the bacterial β and eukaryotic proliferating cell nuclear antigen (PCNA), are multimeric, with a binding cleft on each protomer. Biochemical experiments with bacterial (3, 4) and eukaryotic (5) proteins have suggested that clamps can simultaneously bind multiple DNA polymerases during active DNA synthesis, serving as a molecular toolbelt (6). This multivalency may facilitate rapid polymerase exchange and lesion bypass. However, it remains unclear if and when large multisubunit replicative polymerases can accommodate Y-family polymerases on the clamp. Furthermore, most organisms have multiple Y-family polymerases and many other clamp-binding proteins—at least 10 in Escherichia coli (7) and over 50 in humans (8). It is consequently an open question how the correct polymerase is selected at a DNA lesion (1).To further elucidate the role of processivity clamps in polymerase trafficking, we studied the E. coli replicative polymerase, the polymerase (Pol) III heterotrimer αεθ, and the Y-family Pol IV, which individually bind the dimeric β clamp. Pol IV, encoded by the gene dinB, is widely conserved across the three domains of life, and it is the homolog of human Pol κ (9). In addition to its function in lesion bypass (10), E. coli Pol IV is required for the mechanistically controversial phenomenon of stress-induced mutagenesis (11, 12), which is proposed to occur by its preferential synthesis at double-strand break intermediates (13, 14), and involved in reactive oxygen species-mediated antibiotic lethality by its incorporation of oxidized nucleotides into the genome (15).Interactions of Pol IV with β and their implications for the toolbelt model have generated widespread interest (4, 16). The structure of the C-terminal little finger domain of Pol IV bound to β revealed that Pol IV can simultaneously interact with the cleft and the rim, a secondary site of β near its dimer interface, which positions the Pol IV catalytic domain well away from the DNA running through the center of the clamp (17). Although this potential inactive binding mode for Pol IV has been interpreted as evidence for the toolbelt model, Pol III was shown to contain a second weak CBM in its ε exonuclease subunit (18) in addition to the CBM in its α catalytic subunit that has a strong affinity for the β clamp. A recent study proposed that Pol III would, therefore, occlude Pol IV from clamp binding during replication, only accommodating simultaneous binding after a lesion-induced stall (19).Previous efforts to reconstitute this model system for polymerase exchange have involved stalling Pol III on β at a primer terminus by nucleotide omission to synchronize a population of molecules and simulate a lesion-induced block (3, 4). These studies were not able to resolve an exchange back to Pol III after Pol IV synthesis. To bypass these limitations and elucidate the molecular mechanism of exchange between Pol III and Pol IV, we developed a single-molecule assay to observe the whole TLS reaction, quantifying polymerase exchange and bypass at site-specific DNA lesions. Here, we show that Pol III and Pol IV can simultaneously bind β during active synthesis, enabling rapid lesion bypass, and report a previously unidentified inactive binding mode for Pol III. We also observe that, at high concentrations (corresponding to up-regulated levels during the SOS DNA damage response), Pol IV occupies a secondary contact on β, promoting dissociation of Pol III. These results support a model in which secondary contacts between processivity clamps and Y-family polymerases establish a hierarchy for polymerase selection.     

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.DNA double-strand breaks (DSBs) are toxic lesions that can be repaired by two major pathways in eukaryotes: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (1). Although HR repairs DSBs in a template-dependent, high-fidelity manner, NHEJ functions to ligate DSB ends together using no or very short (1–4 bp) homology. Recently, a new pathway was identified in eukaryotes, which uses microhomologies (MHs) to repair a DSB and does not require the central proteins used in HR (Rad51, Rad52) or NHEJ (Ku70–Ku80) (2–5). In mammalian cells, this pathway of repair is known as alternative end-joining (Alt-EJ) and is often but not always associated with MHs, whereas in budding yeast, the commensurate pathway, MH-mediated end-joining (MMEJ), will typically use 5–25 bp of MH (6, 7). These pathways are associated with genomic rearrangements, and cancer genomes show evidence of MH-mediated rearrangements (8–12). In addition, eukaryotic genomes contain many dispersed repetitive elements that can lead to genome rearrangements when recombination occurs between them (13–16). Therefore, controlling DSB repair in the human genome, which features a variety of repeats, is especially important given the fact that recombination between repetitive elements has been implicated in genomic instability associated with disease (17–20).The original characterization of Alt-EJ in mammalian cells suggested it did not represent a significant DNA repair pathway and only operated in the absence of functional HR and NHEJ pathways. More recent analyses demonstrate a physiological role of Alt-EJ during DNA repair in the presence of active HR and NHEJ pathways (2, 12, 21, 22). Furthermore, examination of I-SceI–induced translocation junctions in mammalian cells revealed the frequent presence of MHs (23, 24). NHEJ-deficient and p53-null mice develop pro–B-cell lymphomas, and nonreciprocal translocations characterized by small MHs are found at their break point junctions (25–28). Similarly, in human cancers, many translocation break point junctions contain MHs, suggesting a role for Alt-EJ in cancer development (29–31) and resistance to chemotherapy and genetic disease (32–36). Hence, the presence of many short repetitive sequences in the human genome is likely to increase rearrangements mediated by MHs following the creation of a DSB.MMEJ is a distinct DSB repair pathway that operates in the presence of functional NHEJ and HR pathways (10, 37). The genetic requirements of MMEJ are being studied in the model eukaryote Saccharomyces cerevisiae and involve components traditionally considered specific to the NHEJ (Pol λ) and HR (Rad1–Rad10, Rad59, and Mre11–Rad50–Xrs2) pathways (4, 5, 10, 38). Although being clearly independent of the central NHEJ factor Ku70–Ku80 heterodimer (10, 37), the involvement of the key HR factor Rad52 in MMEJ remains uncertain. It has been reported that Rad52 is required for MMEJ repair (4, 10, 38), whereas in another assay system Rad52 suppresses MMEJ repair (37). More recently, it has been proposed that the replication protein A (RPA) regulates pathway choice between HR and MMEJ (37). In addition, several models have been proposed that identify specific pathways that may use MHs for the repair of DNA damage (39–41). Despite current advancements in our understanding of MMEJ, the precise involvement of DNA polymerases in supporting the repair of DSBs using MHs remains poorly understood. DNA polymerase λ (also called Pol4 in budding yeast) and its human homolog Pol λ are considered to be the primary candidates for the DNA polymerases working in NHEJ and MMEJ (4, 5, 42–46). Both genetic and biochemical evidence shows that Pol δ is recruited during HR to extend Rad51-dependent recombination intermediates (47–50). Recent analysis using pol32 mutants (5, 10) implicated the Pol32 subunit of Pol δ in MMEJ. Pol32 and Pol31 were also identified as subunits of the DNA polymerase zeta complex (Pol ζ) (51, 52), but previous analysis showed no effect of rev3 mutants in MMEJ (10). REV3 encodes the catalytic subunit of Pol ζ. However, an involvement of Pol δ had not been demonstrated directly before, and it is possible that Pol32 could act in conjunction with yet another DNA polymerase.Here, we report the development of a series of interchromosomal MMEJ assays in diploid S. cerevisiae to assess the mechanisms underlying the repair of DSBs using varying MHs. We focus on diploid cells, as they represent the natural state of budding yeast, which is a diplontic organism (53). The yeast mating-type switching system represents a mechanism to return haploid yeast as efficiently as possible to diploidy (54). Using a combination of genetic, molecular, and in vivo chromatin immunoprecipitation (ChIP) experiments, we provide compelling evidence for a direct involvement of Pol δ in coordinating with Pol λ in MMEJ in budding yeast.