Charly Chahwan

ATM Activation and Its Recruitment to Damaged DNA Require Binding to the C Terminus of Nbs1

ABSTRACT ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-β. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.

Sws1 is a conserved regulator of homologous recombination in eukaryotic cells

Rad52‐dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM‐type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Δ and rqh1Δ mutants and rescues the inviability of srs2Δ rqh1Δ cells. Sws1 functions at an early step of recombination in a pro‐recombinogenic complex with Rlp1 and Rdl1, two RecA‐like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2–RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells.

The Fission Yeast Rad32 (Mre11)-Rad50-Nbs1 Complex Is Required for the S-Phase DNA Damage Checkpoint

ABSTRACT Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G2 DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.

DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication.

Xlf1 Is Required for DNA Repair by Nonhomologous End Joining in Schizosaccharomyces pombe

The accurate repair of DNA double-strand breaks is essential for cell survival and maintenance of genome integrity. Here we describe xlf1+, a gene in the fission yeast Schizosaccharomyces pombe that is required for repair of double-strand breaks by nonhomologous end joining during G1 phase of the cell cycle. Xlf1 is the ortholog of budding yeast Nej1 and human XLF/Cernunnos proteins.

Repression of G1/S transcription is mediated via interaction of the GTB motifs of Nrm1 and Whi5 with Swi6.

ABSTRACT In Saccharomyces cerevisiae, G1/S transcription factors MBF and SBF regulate a large family of genes important for entry to the cell cycle and DNA replication and repair. Their regulation is crucial for cell viability, and it is conserved throughout evolution. MBF and SBF consist of a common component, Swi6, and a DNA-specific binding protein, Mbp1 and Swi4, respectively. Transcriptional repressors bind to and regulate the activity of both transcription factors. Whi5 binds to SBF and represses its activity at the beginning of the G1 phase to prevent early activation. Nrm1 binds to MBF to repress transcription as cells progress through S phase. Here, we describe a protein motif, the GTB motif (for G1/S transcription factor binding), in Nrm1 and Whi5 that is required to bind to the transcription factors. We also identify a region of the carboxy terminus of Swi6 that is required for Nrm1 and Whi5 binding to their target transcription factors and show that mutation of this region overrides the repression of MBF- and SBF-regulated genes by Nrm1 and Whi5. Finally, we show that the GTB motif is the core of a functional module that is necessary and sufficient for targeting of the transcription factors by their cognate repressors.