Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

• Published in: DNA repair Vol. 31; pp. 80 - 90
• Main Authors: Hwang, Bor-Jang, Jin, Jin, Gunther, Randall, Madabushi, Amrita, Shi, Guoli, Wilson, Gerald M., Lu, A.-Lien
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2015
• Subjects: Animals; Binding Sites; Cell Cycle Checkpoints; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cloning, Molecular; DNA binding protein; DNA damage response; DNA glycosylase; DNA Glycosylases - genetics; DNA Glycosylases - metabolism; DNA Repair; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Exonucleases - genetics; Exonucleases - metabolism; Genomic instability; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Mice; Protein Conformation; h coefficient; PCNA; DTT; DNA damage response; 9–1–1; IDC; DNA glycosylase; Rad91−266; DNA repair; RFC; Genomic instability; DDR; EMSA; TBE; G0 or 8-oxoG; MYH; APE1; IDCL; DNA binding protein; h; GST; m; BER; AP; ECL; SDS-PAGE; [P]1/2; Sp; PCR
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2015.05.004

[Display omitted] •Individual Rad9–Rad1–Hus1 (9–1–1) subunits play distinct roles in BER.•The interdomain connecting loop of Hus1 is a key determinant of MYH binding.•The K136A mutant demonstrated that Hus1 binding is uncoupled from MYH activation.•The order of DNA binding affinity is: 91−266–1–1≥Rad91−266•Hus1≫Rad1.•Preferential binding of 9–1–1 to 5′-recessed DNA is independent of clamp loader. 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 Hus1K136A 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, Rad91−266 (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 91−266–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 91−266–1–1 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.