Purification and Characterization of Human DNA Damage Checkpoint Rad Complexes

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2001 Sep 27

PMID 11572977

Citations 69

Authors

V P Bermudez

J Hurwitz

A Sancar

Affiliations

Soon will be listed here.

Abstract

Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.

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References

Kastan M, Lim D . The many substrates and functions of ATM. Nat Rev Mol Cell Biol. 2001; 1(3):179-86. DOI: 10.1038/35043058. View

Shimomura T, Ando S, Matsumoto K, Sugimoto K . Functional and physical interaction between Rad24 and Rfc5 in the yeast checkpoint pathways. Mol Cell Biol. 1998; 18(9):5485-91. PMC: 109133. DOI: 10.1128/MCB.18.9.5485. View

Venclovas C, Thelen M . Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucleic Acids Res. 2000; 28(13):2481-93. PMC: 102700. DOI: 10.1093/nar/28.13.2481. View

Oconnell M, Walworth N, Carr A . The G2-phase DNA-damage checkpoint. Trends Cell Biol. 2000; 10(7):296-303. DOI: 10.1016/s0962-8924(00)01773-6. View

Lydall D, Weinert T . G2/M checkpoint genes of Saccharomyces cerevisiae: further evidence for roles in DNA replication and/or repair. Mol Gen Genet. 1998; 256(6):638-51. DOI: 10.1007/s004380050612. View

Ellison V, Stillman B . Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity. J Biol Chem. 1998; 273(10):5979-87. DOI: 10.1074/jbc.273.10.5979. View

Griffiths D, Barbet N, Mccready S, Lehmann A, Carr A . Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins. EMBO J. 1995; 14(23):5812-23. PMC: 394699. DOI: 10.1002/j.1460-2075.1995.tb00269.x. View

Bessho T, Sancar A . Human DNA damage checkpoint protein hRAD9 is a 3' to 5' exonuclease. J Biol Chem. 2000; 275(11):7451-4. DOI: 10.1074/jbc.275.11.7451. View

St Onge R, Udell C, Casselman R, Davey S . The human G2 checkpoint control protein hRAD9 is a nuclear phosphoprotein that forms complexes with hRAD1 and hHUS1. Mol Biol Cell. 1999; 10(6):1985-95. PMC: 25401. DOI: 10.1091/mbc.10.6.1985. View

10.

Hanna J, Kroll E, Lundblad V, Spencer F . Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Mol Cell Biol. 2001; 21(9):3144-58. PMC: 86942. DOI: 10.1128/MCB.21.9.3144-3158.2001. View

11.

Volkmer E, Karnitz L . Human homologs of Schizosaccharomyces pombe rad1, hus1, and rad9 form a DNA damage-responsive protein complex. J Biol Chem. 1999; 274(2):567-70. DOI: 10.1074/jbc.274.2.567. View

12.

Zhang G, Gibbs E, Kelman Z, ODonnell M, Hurwitz J . Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen. Proc Natl Acad Sci U S A. 1999; 96(5):1869-74. PMC: 26703. DOI: 10.1073/pnas.96.5.1869. View

13.

Waga S, Stillman B . The DNA replication fork in eukaryotic cells. Annu Rev Biochem. 1998; 67:721-51. DOI: 10.1146/annurev.biochem.67.1.721. View

14.

Parker A, Van de Weyer I, Laus M, Oostveen I, Yon J, Verhasselt P . A human homologue of the Schizosaccharomyces pombe rad1+ checkpoint gene encodes an exonuclease. J Biol Chem. 1998; 273(29):18332-9. DOI: 10.1074/jbc.273.29.18332. View

15.

Bao S, Tibbetts R, Brumbaugh K, Fang Y, Richardson D, Ali A . ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses. Nature. 2001; 411(6840):969-74. DOI: 10.1038/35082110. View

16.

Chen M, Lin Y, Lieberman H, Chen G, Lee E . ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation. J Biol Chem. 2001; 276(19):16580-6. DOI: 10.1074/jbc.M008871200. View

17.

Sancar A, Hearst J . Molecular matchmakers. Science. 1993; 259(5100):1415-20. DOI: 10.1126/science.8451638. View

18.

Kostrub C, Knudsen K, Subramani S, Enoch T . Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage. EMBO J. 1998; 17(7):2055-66. PMC: 1170550. DOI: 10.1093/emboj/17.7.2055. View

19.

Chang M, Sasaki H, Campbell M, Kraeft S, Sutherland R, Yang C . HRad17 colocalizes with NHP2L1 in the nucleolus and redistributes after UV irradiation. J Biol Chem. 1999; 274(51):36544-9. DOI: 10.1074/jbc.274.51.36544. View

20.

Kondo T, Matsumoto K, Sugimoto K . Role of a complex containing Rad17, Mec3, and Ddc1 in the yeast DNA damage checkpoint pathway. Mol Cell Biol. 1999; 19(2):1136-43. PMC: 116043. DOI: 10.1128/MCB.19.2.1136. View