第7章 平成22年度～24年度特別研究 : 「地域歴史遺産保全活用教育研究を基軸とした地域歴史文化育成支援拠点の整備」事業

textabstractThe Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 threedimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions

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

SummaryThe 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-BRCT1-BRCT2 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

Critical Functions of Rpa3/Ssb3 in S-Phase DNA Damage Responses in Fission Yeast

Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)–binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11 Rpa1 and Rad22 Rad52 nuclear foci in ssb3D cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks.

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair

Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks▿

Rad3, the Schizosaccharomyces pombe ortholog of human ATR and Saccharomyces cerevisiae Mec1, activates the checkpoint kinase Chk1 in response to DNA double-strand breaks (DSBs). Rad3ATR/Mec1 associates with replication protein A (RPA), which binds single-stranded DNA overhangs formed by DSB resection. In humans and both yeasts, DSBs are initially detected and processed by the Mre11-Rad50-Nbs1Xrs2 (MRN) nucleolytic protein complex in association with the Tel1ATM checkpoint kinase and the Ctp1CtIP/Sae2 DNA-end processing factor; however, in budding yeast, neither Mre11 nuclease activity or Sae2 are required for Mec1 signaling at irreparable DSBs. Here, we investigate the relationship between DNA end processing and the DSB checkpoint response in fission yeast, and we report that Mre11 nuclease activity and Ctp1 are critical for efficient Rad3-to-Chk1 signaling. Moreover, deleting Ctp1 reveals a Tel1-to-Chk1 signaling pathway that bypasses Rad3. This pathway requires Mre11 nuclease activity, the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp complex, and Crb2 checkpoint mediator. Ctp1 negatively regulates this pathway by controlling MRN residency at DSBs. A Tel1-to-Chk1 checkpoint pathway acting at unresected DSBs provides a mechanism for coupling Chk1 activation to the initial detection of DSBs and suggests that ATM may activate Chk1 by both direct and indirect mechanisms in mammalian cells

BRCT Domain Interactions with Phospho-Histone H2A Target Crb2 to Chromatin at Double-Strand Breaks and Maintain the DNA Damage Checkpoint▿

Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3ATR, forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT2) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1ATM and Rad3ATR, although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A (γ-H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT2 domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT2 γ-H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the γ-H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins

Critical Function of γH2A in S-Phase

<div><p>Abstract</p><p>Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in <i>Schizosaccharomyces pombe</i>. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.</p></div

Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast

<div><p>The DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) protects genome integrity by restoring ligatable 5’-phosphate and 3’-hydroxyl termini at single-strand breaks (SSBs). In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR) of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (<i>pnk1Δ</i>) cells of <i>Schizosaccharomyces pombe</i>. Here, we report that <i>pnk1Δ</i> cells have enhanced requirements for Rad3 (ATR/Mec1) and Chk1 checkpoint kinases, and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A (γH2A) at damaged replication forks. The viability of <i>pnk1Δ</i> cells depends on Mre11 and Ctp1 (CtIP/Sae2) double-strand break (DSB) resection proteins, Rad52 DNA strand annealing protein, Mus81-Eme1 Holliday junction resolvase, and Rqh1 (BLM/WRN/Sgs1) DNA helicase. Coupled with increased sister chromatid recombination and Rad52 repair foci in <i>pnk1Δ</i> cells, these findings indicate that lingering SSBs in <i>pnk1Δ</i> cells trigger Rad51-independent homology-directed repair of collapsed replication forks. From these data, we propose models for HDR-mediated tolerance of persistent SSBs with 3’ phosphate in <i>pnk1Δ</i> cells.</p></div