The Mre11 Complex Mediates the S-Phase Checkpoint through an Interaction with Replication Protein A

Abstract

The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.The Mre11/Rad50/Nbs1 complex (MRN) participates in multiple pathways to maintain genome stability (1, 10, 55). In humans, hypomorphic mutations in NBS1 and MRE11 lead to Nijmegen breakage syndrome (NBS) and ataxia-telangiectasialike disorder (ATLD), respectively (7,41,54,64). Both NBS and ATLD patients show developmental defects, immunodeficiency, and a high incidence of cancer, phenotypically similar to the ATM-deficient disorder ataxia-telangiectasia (AT) (53, 60). In addition, cells from NBS and ATLD patients are sensitive to radiation, exhibit genome instability, and have a defective S-phase checkpoint.The phenotypic resemblance of NBS, ATLD, and AT cells implies that MRN and ATM participate in similar biological pathways. This is supported by recent findings that MRN acts both upstream and downstream of ATM to mediate the damage response when double-strand breaks (DSBs) are generated (32). MRN migrates to DSB sites immediately after damage in an ATM-independent manner (44, 47) and is required for ATM activation, especially in response to low doses of ionizing radiation (IR) (6,8,25,35,63). MRN is also a direct substrate of ATM. Multiple ATM phosphorylation sites on Nbs1 have been identified, and these phosphorylation events play important roles in mediating the intra-S and G 2 /M checkpoints, although the mechanism by which Nbs1 phosphorylation regulates these pathways is not well defined (8,22,36,69,75).Mre11 is the core of the MRN complex and interacts with both Rad50 and Nbs1 (24, 34, 62). Mre11 carries 3Ј to 5Ј exonuclease activity and single-strand endonuclease activity and acts on various types of DSB ends and hairpins (49, 61). These nuclease activities are believed to be involved in processing of DNA ends for DSB repair (18,37,50). In multiple organisms, it has been shown that MRN is essential for DNA repair by homologous recombination (27,59,70).The radioresi...

The Basic Cleft of RPA70N Binds Multiple Checkpoint Proteins, Including RAD9, To Regulate ATR Signaling

“…Notably, this region contains the acidic peptide that we used in the NMR experiment. Furthermore, double mutation of D543 and D544 in MRE11 abolished the RPA interaction, impaired the localization of MRE11 to replication centers, and caused a defect in the S-phase checkpoint (41). Our data suggest that this mutant disrupts an MRE11 CRD that interacts with the basic cleft of RPA70N.…”

“…2), a recent publication mapped an RPA binding surface on MRE11 to amino acids 521 to 569 (41). Notably, this region contains the acidic peptide that we used in the NMR experiment.…”

Nucleoside analogs: molecular mechanisms signaling cell death

“…Dysfunction of ATM or the MRN complex subunits results in embryonic lethality in eukaryotes (Xiao and Weaver, 1997;Luo et al, 1999;Zhu et al, 2001a) and hypomorphic mutations are associated with a variety of human disorders, including ataxia-telangiectasia (AT), ataxia-telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS, Carney et al, 1998;Matsuura et al, 1998;Varon et al, 1998;Stewart et al, 1999;Shiloh, 2006), suggesting their involvement during normal DNA replication. At the molecular level, the MRN complex associates with chromatin in an S phasespecific manner (Mirzoeva and Petrini, 2001) and binds with RPA (Robison et al, 2004;Olson et al, 2007). A similar phenomenon is evident in response to stalled replication forks.…”

The RING Finger Protein RNF8 Ubiquitinates Nbs1 to Promote DNA Double-strand Break Repair by Homologous Recombination

“…Cleared cell lysates were then collected for immunoprecipitation and/or subjected to SDS-PAGE as described (47). Immunostaining and immunofluorescence microscopy analysis were performed as described (47,48).…”