Interactions of Human Mismatch Repair Proteins MutSα and MutLα with Proteins of the ATR-Chk1 Pathway

DNA damage response (DDR) activates a complex signaling network that triggers DNA repair, cell cycle arrest, and/or cell death. Depending on the type and severity of DNA lesion, DDR is controlled by "master" regulators including ATM and ATR protein kinases. Cisplatin, a major chemotherapy drug that cross-links DNA, induces ATR-dependent DDR, resulting in apoptosis. However, it is unclear how ATR is activated. To identify the key regulators of ATR, we analyzed the proteins that associate with ATR after cisplatin treatment by blue native-PAGE and co-immunoprecipitation. The mismatch repair protein hMSH2 was found to be a major ATR-binding protein.Functionally, ATR activation and its recruitment to nuclear foci during cisplatin treatment were attenuated, and DNA damage signaling, involving Chk2, p53, and PUMA-␣, was suppressed in hMSH2-deficient cells. ATR activation induced by the DNA methylating agent N-methyl-N-nitrosourea was also shown to be hMSH2-dependent. Intriguingly, hMSH2-mediated ATR recruitment and activation appeared independent of replication protein A, Rad17, and the Rad9-Hus1-Rad1 protein complex. Together the results support a hMSH2-dependent pathway of ATR activation and downstream Chk2/p53 signaling. DNA damage response (DDR)3 is essential for the maintenance of the integrity of the genome (1-5). As a complex multistep process, DDR involves the recognition of DNA damage, activation of DNA damage-responsive protein kinases, signal amplification by downstream protein kinases, and activation of the effector proteins that trigger various cellular processes. At low levels of DNA damage, activation of DDR results in cell cycle arrest and DNA repair. However, at higher doses, DDR signaling frequently results in cell death by apoptosis (1-5). The phosphoinositide 3-kinase-related protein kinases, ATM (ataxiatelangiectasia mutated) and ATR (ATM-and Rad3-related), are the key regulators of DDR (1-8). Once activated, ATM and ATR regulate an array of substrates including Chk1 and Chk2, which culminate in DNA repair, cell cycle arrest, and/or apoptosis. Although ATM is generally activated by doublestranded DNA breaks, ATR activation can result from different types of DNA lesion including single-stranded breaks, replication stress, base adducts, and DNA cross-links (8, 9). In the canonical model, ATR activation involves the recruitment of the ATR-ATRIP (ATR-interacting protein) and the Rad9-Hus1-Rad1 (9-1-1) protein complexes to the DNA damage site via replication protein A (RPA). As a result, the 9-1-1 complex brings topoisomerase-binding protein-1 (TopBP1, an ATR activator) close to ATR for ATR activation (1,5,8). However, alternative pathways of ATR activation may exist. For example, mismatch repair (MMR) proteins have been implicated in ATR activation under certain experimental conditions (10 -12).The MMR system is important for DNA replication and repair (13-18). In mammalian cells, MMR is composed of five MutS homologues (MSH) and four MutL homologue proteins. The function of MMR is to first recognize D...

Section: Hmsh2 In Atr Activationmentioning

confidence: 61%

“…MMR proteins were shown to directly interact with ATR, TopBP1, and Chk1 (12). Our present study demonstrates ATR/hMSH2 interaction during cisplatin-induced DNA damage by both BN-PAGE and co-IP analysis (Fig.…”

Section: Hmsh2 In Atr Activationmentioning

confidence: 72%

hMSH2 Recruits ATR to DNA Damage Sites for Activation during DNA Damage-induced Apoptosis

Pabla

McIlhatton

et al. 2011

“…In vitro studies suggest that ATR and ATRIP can be recruited to sites of O 6 -meG:T mispairs in a pathway dependent on MutS␣ and MutL␣ (53). Also, it has been reported that MutS␣ and MutL␣ serve as a scaffold for recruiting the checkpoint proteins ATR, TopBP1, and Chk1 after treatment with the S n 1 alkylating agent MNNG (54).…”

Section: Discussionmentioning

confidence: 99%

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

Geng

Sakato

DeRocco

et al. 2012

Background: MutS␣, a heterodimer of MSH2 and MSH6, is essential for DNA mismatch repair. Results: hMsh2 G674A -hMSH6 wt and hMSH2 wt -hMSH6 T1219D mutant proteins fail to efficiently license mismatch-provoked, nick-directed excision. Conclusion: Different defects underlie the apparently similar repair deficiency of these two mutants. Significance: Findings provide new insights into the mechanism of mismatch repair with implications for cancer predisposition and the apoptotic response to DNA damaging agents.

“…2) Recruitment by Repair Proteins. It has been reported that the nucleotide excision repair protein XPA and the mismatch repair protein MSH2 bind to the respective damage/mismatch sites and recruit ATR (Mec1 in budding yeast) to chromatin, leading to its activation (36,47,48). 3) Recruitment by the Repair Gap.…”

Section: Discussionmentioning

confidence: 99%

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

Lindsey-Boltz

Kemp

Reardon

et al. 2014

Self Cite

Background: Nucleotide excision repair and the ATR-mediated DNA damage checkpoint responses are genetically coupled. Results: We have analyzed the basic steps of ATR activation in a biochemically defined system. Conclusion: ATR signaling requires enlargement of the DNA excision gap by EXO1. Significance: The six excision repair factors, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of proteins for ATR-activation upon UV-induced DNA damage.