Checkpoint Kinase 2 (Chk2) Inhibits the Activity of the Cdc45/MCM2-7/GINS (CMG) Replicative Helicase Complex

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2012 Aug 3

PMID 22853956

Citations 22

Authors

Ivar Ilves

Nele Tamberg

Michael R Botchan

Affiliations

Soon will be listed here.

Abstract

The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. In this report we show that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila melanogaster CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and we suggest that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage.

Citing Articles

Unwinding Helicase MCM Functionality for Diagnosis and Therapeutics of Replication Abnormalities Associated with Cancer: A Review.

Radhakrishnan A, Gangopadhyay R, Sharma C, Kapardar R, Sharma N, Srivastav R Mol Diagn Ther. 2024; 28(3):249-264.

PMID: 38530633 DOI: 10.1007/s40291-024-00701-5.

Distinct functions of EHMT1 and EHMT2 in cancer chemotherapy and immunotherapy.

Kang Z, Fu P, Ma H, Li T, Lu K, Liu J bioRxiv. 2023; .

PMID: 37873068 PMC: 10592889. DOI: 10.1101/2023.10.03.560719.

Rad53 arrests leading and lagging strand DNA synthesis via distinct mechanisms in response to DNA replication stress.

He R, Zhang Z Bioessays. 2022; 44(9):e2200061.

PMID: 35778827 PMC: 10316031. DOI: 10.1002/bies.202200061.

Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks.

Devbhandari S, Remus D Nat Struct Mol Biol. 2020; 27(5):461-471.

PMID: 32341532 PMC: 7225081. DOI: 10.1038/s41594-020-0407-7.

Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling.

Can G, Kauerhof A, Macak D, Zegerman P Mol Cell. 2019; 73(3):562-573.e3.

PMID: 30595439 PMC: 6375734. DOI: 10.1016/j.molcel.2018.11.025.

References

Sheu Y, Stillman B . The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature. 2010; 463(7277):113-7. PMC: 2805463. DOI: 10.1038/nature08647. View

Stracker T, Usui T, Petrini J . Taking the time to make important decisions: the checkpoint effector kinases Chk1 and Chk2 and the DNA damage response. DNA Repair (Amst). 2009; 8(9):1047-54. PMC: 2725228. DOI: 10.1016/j.dnarep.2009.04.012. View

Gambus A, Jones R, Sanchez-Diaz A, Kanemaki M, van Deursen F, Edmondson R . GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat Cell Biol. 2006; 8(4):358-66. DOI: 10.1038/ncb1382. View

Botchan M, Berger J . DNA replication: making two forks from one prereplication complex. Mol Cell. 2010; 40(6):860-1. PMC: 3023982. DOI: 10.1016/j.molcel.2010.12.014. View

Bahassi E, Robbins S, Yin M, Boivin G, Kuiper R, van Steeg H . Mice with the CHEK2*1100delC SNP are predisposed to cancer with a strong gender bias. Proc Natl Acad Sci U S A. 2009; 106(40):17111-6. PMC: 2761365. DOI: 10.1073/pnas.0909237106. View

Remus D, Beuron F, Tolun G, Griffith J, Morris E, Diffley J . Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell. 2009; 139(4):719-30. PMC: 2804858. DOI: 10.1016/j.cell.2009.10.015. View

Lin D, Aggarwal P, Diehl J . Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2-7 complex. Proc Natl Acad Sci U S A. 2008; 105(23):8079-84. PMC: 2430362. DOI: 10.1073/pnas.0800077105. View

Piccoli G, Katou Y, Itoh T, Nakato R, Shirahige K, Labib K . Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases. Mol Cell. 2012; 45(5):696-704. DOI: 10.1016/j.molcel.2012.01.007. View

Choi J, Lim H, Kim J, Song O, Cho Y . Crystal structure of the human GINS complex. Genes Dev. 2007; 21(11):1316-21. PMC: 1877744. DOI: 10.1101/gad.1548107. View

10.

Painter R, Young B . X-ray-induced inhibition of DNA synthesis in Chinese hamster ovary, human HeLa, and Mouse L cells. Radiat Res. 1975; 64(3):648-56. View

11.

Li J, Deng M, Wei Q, Liu T, Tong X, Ye X . Phosphorylation of MCM3 protein by cyclin E/cyclin-dependent kinase 2 (Cdk2) regulates its function in cell cycle. J Biol Chem. 2011; 286(46):39776-85. PMC: 3220541. DOI: 10.1074/jbc.M111.226464. View

12.

Yoo H, Shevchenko A, Shevchenko A, Dunphy W . Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses. J Biol Chem. 2004; 279(51):53353-64. DOI: 10.1074/jbc.M408026200. View

13.

Painter R, Young B . Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad Sci U S A. 1980; 77(12):7315-7. PMC: 350493. DOI: 10.1073/pnas.77.12.7315. View

14.

Costa A, Ilves I, Tamberg N, Petojevic T, Nogales E, Botchan M . The structural basis for MCM2-7 helicase activation by GINS and Cdc45. Nat Struct Mol Biol. 2011; 18(4):471-7. PMC: 4184033. DOI: 10.1038/nsmb.2004. View

15.

Brodsky M, Weinert B, Tsang G, Rong Y, McGinnis N, Golic K . Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol Cell Biol. 2004; 24(3):1219-31. PMC: 321428. DOI: 10.1128/MCB.24.3.1219-1231.2004. View

16.

Schultz K, Friesen P . Baculovirus DNA replication-specific expression factors trigger apoptosis and shutoff of host protein synthesis during infection. J Virol. 2009; 83(21):11123-32. PMC: 2772793. DOI: 10.1128/JVI.01199-09. View

17.

Kwak E, Kim S, Zhang J, Cardiff R, Schmidt E, Haber D . Mammary tumorigenesis following transgenic expression of a dominant negative CHK2 mutant. Cancer Res. 2006; 66(4):1923-8. PMC: 2865171. DOI: 10.1158/0008-5472.CAN-05-1237. View

18.

Matsuoka S, Ballif B, Smogorzewska A, McDonald 3rd E, Hurov K, Luo J . ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science. 2007; 316(5828):1160-6. DOI: 10.1126/science.1140321. View

19.

Holt L, Tuch B, Villen J, Johnson A, Gygi S, Morgan D . Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science. 2009; 325(5948):1682-6. PMC: 2813701. DOI: 10.1126/science.1172867. View

20.

Peters M, DeLuca C, Hirao A, Stambolic V, Potter J, Zhou L . Chk2 regulates irradiation-induced, p53-mediated apoptosis in Drosophila. Proc Natl Acad Sci U S A. 2002; 99(17):11305-10. PMC: 123252. DOI: 10.1073/pnas.172382899. View