title: Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA.
abstract: Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Bor-Jang
authors.initials: BJ
authors.lastname: Hwang
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Jin
authors.initials: J
authors.lastname: Jin
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Randall
authors.initials: R
authors.lastname: Gunther
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States; Department of Natural and Physical Sciences, Life Sciences Institute; Baltimore City Community College, Baltimore, MD 21201, United States.
authors.forename: Amrita
authors.initials: A
authors.lastname: Madabushi
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States; University of Maryland School of Nursing, Baltimore, MD 21201, United States.
authors.forename: Guoli
authors.initials: G
authors.lastname: Shi
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States; Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Wilson
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States; Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, United States. Electronic address: alu-chang@som.umaryland.edu.
authors.forename: A-Lien
authors.initials: AL
authors.lastname: Lu
mesh_terms: D000818:Animals; D001665:Binding Sites; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D045647:DNA Glycosylases / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005092:Exonucleases / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011487:Protein Conformation
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C549605:HUS1 protein, human; C069930:rad9 protein; D005982:Glutathione Transferase; D005092:Exonucleases; C113574:Rad1 protein, human; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase
keywords: DNA binding protein; DNA damage response; DNA glycosylase; DNA repair; Genomic instability