Jessica S. Williams

Mre11 Dimers Coordinate DNA End Bridging and Nuclease Processing in Double-Strand-Break Repair

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).

Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template.

The Mre11-Rad50-Nbs1 (MRN) complex is providing paradigm-shifting results of exceptional biomedical interest. MRN is among the earliest respondents to DNA double-strand breaks (DSBs), and MRN mutations cause the human cancer predisposition diseases Nijmegen breakage syndrome and ataxia telangiectasia-like disorder (ATLD). MRNs 3-protein multidomain composition promotes its central architectural, structural, enzymatic, sensing, and signaling functions in DSB responses. To organize the MRN complex, the Mre11 exonuclease directly binds Nbs1, DNA, and Rad50. Rad50, a structural maintenance of chromosome (SMC) related protein, employs its ATP-binding cassette (ABC) ATPase, Zn hook, and coiled coils to bridge DSBs and facilitate DNA end processing by Mre11. Contributing to MRN regulatory roles, Nbs1 harbors N-terminal phosphopeptide interacting FHA and BRCT domains, as well as C-terminal ataxia telangiectasia mutated (ATM) kinase and Mre11 interaction domains. Current emerging structural and biological evidence suggests that MRN has 3 coupled critical roles in DSB sensing, stabilization, signaling, and effector scaffolding: (1) expeditious establishment of protein--nucleic acid tethering scaffolds for the recognition and stabilization of DSBs; (2) initiation of DSB sensing, cell-cycle checkpoint signaling cascades, and establishment of epigenetic marks via the ATM kinase; and (3) functional regulation of chromatin remodeling in the vicinity of a DSB.

Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT(1)-BRCT(2) domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

Mutagenic Processing of Ribonucleotides in DNA by Yeast Topoisomerase I

An enzyme that removes supercoils from DNA can cause mutations when RNA bases accidentally get incorporated into the DNA. The ribonuclease (RNase) H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides [ribonucleoside monophosphates (rNMPs)] from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMP-associated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease.

Fission Yeast Scm3 Mediates Stable Assembly of Cnp1 (CENP-A) into Centromeric Chromatin

Mis16 and Mis18 are subunits of a protein complex required for incorporation of the histone H3 variant CenH3 (Cnp1/CENP-A) into centromeric chromatin in Schizosaccharomyces pombe and mammals. How the Mis16-Mis18 complex performs this function is unknown. Here, we report that the Mis16-Mis18 complex is required for centromere localization of Scm3(Sp), a Cnp1-binding protein related to Saccharomyces cerevisiae Scm3. Scm3(Sp) is required for centromeric localization of Cnp1, while Scm3(Sp) localizes at centromeres independently of Cnp1. Like the Mis16-Mis18 complex but unlike Cnp1, Scm3(Sp) dissociates from centromeres during mitosis. Inactivation of Scm3(Sp) or Mis18 increases centromere localization of histones H3 and H2A/H2B, which are largely absent from centromeres in wild-type cells. Whereas S. cerevisiae Scm3 is proposed to replace histone H2A/H2B in centromeric nucleosomes, the dynamic behavior of S. pombe Scm3 suggests that it acts as a Cnp1 assembly/maintenance factor that directly mediates the stable deposition of Cnp1 into centromeric chromatin.

Ribonucleotides Are Signals for Mismatch Repair of Leading-Strand Replication Errors

To maintain genome stability, mismatch repair of nuclear DNA replication errors must be directed to the nascent strand, likely by DNA ends and PCNA. Here we show that the efficiency of mismatch repair in Saccharomyces cerevisiae is reduced by inactivating RNase H2, which nicks DNA containing ribonucleotides incorporated during replication. In strains encoding mutator polymerases, this reduction is preferential for repair of mismatches made by leading-strand DNA polymerase ε as compared to lagging-strand DNA polymerase δ. The results suggest that RNase-H2-dependent processing of ribonucleotides transiently present in DNA after replication may direct mismatch repair to the continuously replicated nascent leading strand.

ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair

The Rad50 ABC–ATPase complex with Mre11 nuclease is essential for dsDNA break repair, telomere maintenance and ataxia telangiectasia–mutated kinase checkpoint signaling. How Rad50 affects Mre11 functions and how ABC–ATPases communicate nucleotide binding and ligand states across long distances and among protein partners are questions that have remained obscure. Here, structures of Mre11–Rad50 complexes define the Mre11 2-helix Rad50 binding domain (RBD) that forms a four-helix interface with Rad50 coiled coils adjoining the ATPase core. Newly identified effector and basic-switch helix motifs extend the ABC–ATPase signature motif to link ATP-driven Rad50 movements to coiled coils binding Mre11, implying an ~30-Å pull on the linker to the nuclease domain. Both RBD and basic-switch mutations cause clastogen sensitivity. Our new results characterize flexible ATP-dependent Mre11 regulation, defects in cancer-linked RBD mutations, conserved superfamily basic switches and motifs effecting ATP-driven conformational change, and they provide a unified comprehension of ABC–ATPase activities.

Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.

Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5′ DNA end–mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair–deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5′ DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.

Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to >95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.

γH2A binds Brc1 to maintain genome integrity during S‐phase

ATMTel1 and ATRRad3 checkpoint kinases phosphorylate the C‐terminus of histone H2AX (H2A in yeasts) in chromatin flanking DNA damage, establishing a recruitment platform for checkpoint and repair proteins. Phospho‐H2A/X (γH2A/X)‐binding proteins at double‐strand breaks (DSBs) have been characterized, but those required for replication stress responses are unknown. Here, we present genetic, biochemical, small angle X‐ray scattering (SAXS), and X‐ray structural studies of the Schizosaccharomyces pombe Brc1, a 6‐BRCT‐domain protein that is structurally related to Saccharomyces cerevisiae Rtt107 and mammalian PTIP. Brc1 binds γH2A to form spontaneous and DNA damage‐induced nuclear foci. Spontaneous Brc1 foci colocalize with ribosomal DNA repeats, a region prone to fork pausing and genomic instability, whereas DNA damage‐induced Brc1 foci colocalize with DSB response factors. γH2A binding is critical for Brc1 function. The 1.45 Å resolution crystal structure of Brc1–γH2A complex shows how variable BRCT insertion loops sculpt tandem‐BRCT phosphoprotein‐binding pockets to facilitate unique phosphoprotein‐interaction specificities, and unveils an acidic DNA‐mimicking Brc1 surface. From these results, Brc1 docking to γH2A emerges as a critical chromatin‐specific response to replication‐associated DNA damage.