Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage

Mammalian cells respond to DNA double-strand breaks (DSBs) by recruiting DNA repair and cell-cycle checkpoint proteins to such sites. Central to these DNA damage response (DDR) events is the DNA damage mediator protein MDC1. MDC1 interacts with several DDR proteins, including the MRE11–RAD50–NBS1 (MRN) complex. Here, we show that MDC1 is phosphorylated on a cluster of conserved repeat motifs by casein kinase 2 (CK2). Moreover, we establish that this phosphorylation of MDC1 promotes direct, phosphorylation-dependent interactions with NBS1 in a manner that requires the closely apposed FHA and twin BRCT domains in the amino terminus of NBS1. Finally, we show that these CK2-targeted motifs in MDC1 are required to mediate NBS1 association with chromatin-flanking sites of unrepaired DSBs. These findings provide a molecular explanation for the MDC1–MRN interaction and yield insights into how MDC1 coordinates the focal assembly and activation of several DDR factors in response to DNA damage

NBS1 Heterozygosity and Cancer Risk

Biallelic mutations in the NBS1 gene are responsible for the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by chromosome instability and hypersensitivity to ionising radiation (IR). Epidemiological data evidence that the NBS1 gene can be considered a susceptibility factor for cancer development, as demonstrated by the fact that almost 40% of NBS patients have developed a malignancy before the age of 21. Interestingly, also NBS1 heterozygotes, which are clinically asymptomatic, display an elevated risk to develop some types of malignant tumours, especially breast, prostate and colorectal cancers, lymphoblastic leukaemia, and non-Hodgkin’s lymphoma (NHL). So far, nine mutations in the NBS1 gene have been found, at the heterozygous state, in cancer patients. Among them, the 657del5, the I171V and the R215W mutations are the most frequently described. The pathogenicity of these mutations is presumably connected with their occurrence in the highly conserved BRCT tandem domains of the NBS1 protein, which are present in a large superfamily of proteins, and are recognized as major mediators of processes related to cell-cycle checkpoint and DNA repair

Pch2 Acts through Xrs2 and Tel1/ATM to Modulate Interhomolog Bias and Checkpoint Function during Meiosis

Proper segregation of chromosomes during meiosis requires the formation and repair of double-strand breaks (DSBs) to form crossovers. Repair is biased toward using the homolog as a substrate rather than the sister chromatid. Pch2 is a conserved member of the AAA+-ATPase family of proteins and is implicated in a wide range of meiosis-specific processes including the recombination checkpoint, maturation of the chromosome axis, crossover control, and synapsis. We demonstrate a role for Pch2 in promoting and regulating interhomolog bias and the meiotic recombination checkpoint in response to unprocessed DSBs through the activation of axial proteins Hop1 and Mek1 in budding yeast. We show that Pch2 physically interacts with the putative BRCT repeats in the N-terminal region of Xrs2, a member of the MRX complex that acts at sites of unprocessed DSBs. Pch2, Xrs2, and the ATM ortholog Tel1 function in the same pathway leading to the phosphorylation of Hop1, independent of Rad17 and the ATR ortholog Mec1, which respond to the presence of single-stranded DNA. An N-terminal deletion of Xrs2 recapitulates the pch2Δ phenotypes for signaling unresected breaks. We propose that interaction with Xrs2 may enable Pch2 to remodel chromosome structure adjacent to the site of a DSB and thereby promote accessibility of Hop1 to the Tel1 kinase. In addition, Xrs2, like Pch2, is required for checkpoint-mediated delay conferred by the failure to synapse chromosomes

14-3-3 Proteins, FHA Domains and BRCT Domains in the DNA Damage Response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G2/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest

Nijmegen breakage syndrome (NBS)

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive syndrome of chromosomal instability mainly characterized by microcephaly at birth, combined immunodeficiency and predisposition to malignancies. Due to a founder mutation in the underlying NBN gene (c.657_661del5) the disease is encountered most frequently among Slavic populations. The principal clinical manifestations of the syndrome are: microcephaly, present at birth and progressive with age, dysmorphic facial features, mild growth retardation, mild-to-moderate intellectual disability, and, in females, hypergonadotropic hypogonadism. Combined cellular and humoral immunodeficiency with recurrent sinopulmonary infections, a strong predisposition to develop malignancies (predominantly of lymphoid origin) and radiosensitivity are other integral manifestations of the syndrome. The NBN gene codes for nibrin which, as part of a DNA repair complex, plays a critical nuclear role wherever double-stranded DNA ends occur, either physiologically or as a result of mutagenic exposure. Laboratory findings include: (1) spontaneous chromosomal breakage in peripheral T lymphocytes with rearrangements preferentially involving chromosomes 7 and 14, (2) sensitivity to ionizing radiation or radiomimetics as demonstrated in vitro by cytogenetic methods or by colony survival assay, (3) radioresistant DNA synthesis, (4) biallelic hypomorphic mutations in the NBN gene, and (5) absence of full-length nibrin protein. Microcephaly and immunodeficiency are common to DNA ligase IV deficiency (LIG4 syndrome) and severe combined immunodeficiency with microcephaly, growth retardation, and sensitivity to ionizing radiation due to NHEJ1 deficiency (NHEJ1 syndrome). In fact, NBS was most commonly confused with Fanconi anaemia and LIG4 syndrome. Genetic counselling should inform parents of an affected child of the 25% risk for further children to be affected. Prenatal molecular genetic diagnosis is possible if disease-causing mutations in both alleles of the NBN gene are known. No specific therapy is available for NBS, however, hematopoietic stem cell transplantation may be one option for some patients. Prognosis is generally poor due to the extremely high rate of malignancies

Characterization of the novel endonuclease Sae2 involved in DNA end processing

textAt the very center of sexual reproduction is meiosis. During meiosis, the formation of meiotic Double-Strand-Breaks (DBSs) and their repair by homologous recombination are widely conserved events occurring among most eukaryote species. Meiosis-specific DSB formation requires at least nine proteins (Spo11, Ski8, Rec102, Rec104, Mei4, Mer2, Rec114, Mre11/Rad50/Xrs2) in S. cerevisiae, and the resection of the DSB ends requires additional four proteins (Mre11/Rad50/Xrs2, and Sae2). Spo11 has been identified as the catalytic component of this DSB-initiating complex. However, the roles played by the majority of these proteins are not clear. I have purified the recombinant Spo11/Ski8/Rec102/Rec104 complex, characterized its DNA binding ability as well as its cleavage activity on supercoiled plasmid DNA. Sae2 functions in both meiotic and mitotic repair of DNA double-strand breaks (DSBs) in S. cerevisiae. In vivo experiments have shown that Sae2 collaborates with the Mre11/Rad50/Xrs2 (MRX) complex in DNA end processing. Our laboratory previously showed that recombinant Sae2 exhibits endonuclease activity on single-stranded DNA and single-strand/double-strand DNA junctions using purified proteins in vitro. The MRX complex stimulates Sae2 endonuclease activity on single-stranded DNA close to single-strand/double-strand junctions, through its endonucleolytic activity. However, Sae2 contains no conserved typical nuclease domain, and it only shares very limited homology with its human functional counterpart CtIP. To characterize Sae2 and the active sites responsible for its nuclease activity, I used partial proteolysis and site-directed mutagenesis to analyze the protein. Biochemical assays in vitro show that acidic residues in the central domain play an important role in Sae2 endonuclease activity. Sae2 has also been shown to be phosphorylated by CDK (Cyclin-Dependent Kinase) during the S and G2 phases of the cell cycle, as well as by Tel1/Mec1 upon DNA damage. These modifications are essential for the function of Sae2 in DNA repair, but the function of these modifications are not clear. I have demonstrated that, in the presence of MRX, Sae2 (5D/S267E) mimicking constitutive phosphorylation by CDK and Mec1/Tel1 can assist the 5’ to 3’ exonuclease Exo1 significantly in 5’ end resection by suppressing the inhibitory effect of Ku. These results suggest that Sae2 is a critical switching protein which determines the choice between HR and NHEJ in yeast cells upon DNA damage.Cellular and Molecular Biolog

Étude du rôle de la phosphorylation du complexe Mre11-Rad50-Xrs2 dans le maintien de l'intégrité génomique

L'ADN de chaque cellule est constamment soumis à des stress pouvant compromettre son intégrité. Les bris double-brins sont probablement les dommages les plus nocifs pour la cellule et peuvent être des sources de réarrangements chromosomiques majeurs et mener au cancer s’ils sont mal réparés. La recombinaison homologue et la jonction d’extrémités non-homologues (JENH) sont deux voies fondamentalement différentes utilisées pour réparer ce type de dommage. Or, les mécanismes régulant le choix entre ces deux voies pour la réparation des bris double-brins demeurent nébuleux. Le complexe Mre11-Rad50-Xrs2 (MRX) est le premier acteur à être recruté à ce type de bris où il contribue à la réparation par recombinaison homologue ou JENH. À l’intersection de ces deux voies, il est donc idéalement placé pour orienter le choix de réparation. Ce mémoire met en lumière deux systèmes distincts de phosphorylation du complexe MRX régulant spécifiquement le JENH. L’un dépend de la progression du cycle cellulaire et inhibe le JENH, tandis que l’autre requiert la présence de dommages à l’ADN et est nécessaire au JENH. Ensembles, nos résultats suggèrent que le complexe MRX intègre différents phospho-stimuli pour réguler le choix de la voie de réparation.The genome of every cell is constantly subjected to stresses that could compromise its integrity. DNA double-strand breaks (DSB) are amongst the most damaging events for a cell and can lead to gross chromosomal rearrangements, cell death and cancer if improperly repaired. Homologous recombination and non-homologous end joining (NHEJ) are the main repair pathways responsible for the repair of DSBs. However, the mechanistic basis of both pathways is fundamentally different and the regulation of the choice between both for the repair of DSBs remains largely misunderstood. The Mre11-Rad50-Xrs2 (MRX) complex acts as a DSB first responder and contributes to repair by both homologous recombination and NHEJ. Being at the crossroads of both DSB repair pathways, the MRX complex is therefore in a convenient position to influence the repair choice. This thesis unravels two distinct phosphorylation systems modifying the MRX complex and specifically regulating repair by NHEJ. The first relies on cell cycle progression and inhibits NHEJ, while the second requires the presence of DNA damage and is necessary for efficient NHEJ. Together, our results suggest a model in which the MRX complex would act as an integrator of phospho-stimuli in order to regulate the DSB repair pathway choice

DNA Damage Response to Lesions Involving Both Strands of the Double-helix

DNA damage response is vital to genome maintenance, cell survival and successful transmission of genetic information to daughter cells. This response is extremely important since DNA is subject to damage daily either by endogenous metabolic errors and byproducts or by exposure to genotoxic agents. Different types of lesions are formed as a result of such insults to the DNA; the most toxic of such lesions are those that affect both strands of the double-helix. During my dissertation work, I studied cellular response to DNA lesions such as double-strand breaks and interstrand crosslinks using the model system Drosophila melanogaster. Double-strand breaks are repaired primarily by two mechanisms: homology mediated repair (HR) and nonhomologous end joining (NHEJ). Here I discuss the importance of homology mediated repair by studying repair defects in mutants defective for either of the two genes: 1) nbs gene encodes for the protein Nibrin, which is part of a well characterized protein complex MRN, comprising two other proteins Mre11 and Rad50 2) okra encodes the Drosophila homolog of the Rad54 protein. While the MRN complex is hypothesized to be required during early steps of HR such as break resection, Rad54 is believed to be involved in chromatin remodeling and facilitating the role of the strand invasion protein, Rad51. I have addressed several questions here about the function of MRN in responding to double-strand breaks, using mutations in the nbs gene. Since the NBS protein is known to target the MRN complex to the nucleus, study of NBS in isolation should be reflective of the nuclear function of the MRN complex. The requirement of MRN for NHEJ and /or HR appears to differ in different organism. I found that Drosophila NBS is required for HR and not NHEJ. In addition, I found that in contrast to other studies, MRN may function in late steps of HR, post break resection in Drosophila. Study of defects in responding to DNA damage, specifically double-strand breaks (DSBs), in haploinsufficient nbs mutant backgrounds provided valuable clues into underlying molecular mechanisms that lead to carcinogenesis in human carriers of nbs mutation. I tested to verify if DmRad54 is functionally conserved. This study showed that not only does DmRad54 facilitate DmRad51 function during first round of strand invasion, but it is also required multiple times while repairing the break, during the several rounds of strand invasion and synthesis that is characteristic of HR in pre-meiotic germline cells in Drosophila. The second type of toxic lesion discussed here are the interstrand crosslinks (ICLs). Multiple repair mechanisms integrate to repair interstrand crosslinks in the bacteria Escherichia coli and the budding yeast Saccharomyces cerevisiae. Nucleotide excision repair (NER) and HR proteins are required for ICL repair, among others. Also, since DSB intermediates are formed while resolving ICLs, HR proteins seem to be integral in responding to crosslinks. I tested mutants defective in two genes, mus301 and mus302, both of which are hypersensitive to crosslinking agents, for defects in DSB repair. I found that while mus302 mutants, which have previously been implicated in NER, can repair double-strand breaks normally; mus301 mutants are severely defective in HR, when the only available homologous template for repair is the sister chromatid

The Mre11-Rad50-Xrs2 Complex in the DNA Damage Response

DNA is continuously subjected to various types of damage during normal cellular metabolism. Among these, a DNA double-strand break (DSB) is one of the most cytotoxic lesions, and can lead to genomic instability or cell death if misrepaired or left unrepaired. The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DNA damage through its structural, enzymatic, and signaling roles. It senses DSBs and is essential for both of the two major repair mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). In addition, the complex tethers DNA ends, activates Tel1/ATM kinase, resolves hairpin capped DNA ends and maintains telomere homeostasis. Although significant progress has been made in characterizing the complex, many questions regarding the precise mechanism of how this highly conserved, multifunctional complex manages its various activities in chromosome metabolism remain to be solved. The overarching focus of this thesis is to further expand our understanding of the molecular mechanism and regulation of the MRX complex. Specifically, the contributions of Xrs2, Tel1, and Mre11 3’-5’ dsDNA exonuclease in the multiple roles of the MRX complex are examined. Xrs2/Nbs1, the eukaryotic-specific component of the complex, is required for the nuclear transport of Mre11 and Rad50 and harbors several protein-interacting domains. In order to define the role of Xrs2 as a component of the MRX complex once inside the nucleus, we fused a nuclear localization signal (NLS) to the C terminus of Mre11 and assayed for complementation of xrs2Δ defects. We found that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we showed that the MR complex has the equivalent activity to MRX in cleavage of protein-blocked DNA ends. Although Xrs2 physically interacts with Sae2, end resection in its absence remained Sae2 dependent in vivo and in vitro. MRE11-NLS was unable to rescue the xrs2Δ defects in Tel1 kinase signaling and NHEJ, consistent with the role of Xrs2 as a chaperone and adaptor protein coordinating interactions between the MR and other repair proteins. To further characterize the role of Xrs2 in Tel1 activation, we fused the Tel1 interaction domain of Xrs2 to Mre11-NLS (Mre11-NLS-TID). Mre11-NLS-TID was sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells, indicating that Tel1 recruitment and activation are separate functions of the MRX complex. Unexpectedly, we found a role for Tel1 in stabilizing Mre11-DNA association independently of its kinase activity. This stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Moreover, while nuclear-localized MR complex is sufficient for HR without Xrs2, MR is insufficient for DNA tethering, stalled replication fork stability, and suppression of chromosomal rearrangements. Enforcing Tel1 recruitment to the MR complex fully rescued these defects, highlighting the important roles for Xrs2 and Tel1 in stabilizing the MR complex to prevent replication fork collapse and genomic instability. Lastly, in order to decipher the functional significance of the Mre11 3’-5’ dsDNA exonuclease activity in DSB repair, mre11 mutant alleles reported to be proficient endonuclease and deficient exonuclease were analyzed in vivo and in vitro. Although we did not observe a clear separation of the nuclease activities in vitro, our genetic analysis of the mutant allele is consistent with the current two-stepped, bidirectional model of end resection