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The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates NEIL1 glycosylase.

ABSTRACT: The checkpoint protein Rad9/Rad1/Hus1 heterotrimer (the 9-1-1 complex) is structurally similar to the proliferating cell nuclear antigen sliding clamp and has been proposed to sense DNA damage that leads to cell cycle arrest or apoptosis. Human (h) NEIL1 DNA glycosylase, an ortholog of bacterial Nei/Fpg, is involved in repairing oxidatively damaged DNA bases. In this study, we show that hNEIL1 interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. Residues 290-350 of hNEIL1 are important for the 9-1-1 association. A significant fraction of the hNEIL1 nuclear foci co-localize with hRad9 foci in hydrogen peroxide treated cells. Human NEIL1 DNA glycosylase activity is significantly stimulated by hHus1, hRad1, hRad9 separately and the 9-1-1 complex. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of base excision repair.

SUBMITTER: Guan X

PROVIDER: S-EPMC1885643 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates NEIL1 glycosylase.

Guan Xin X Bai Haibo H Shi Guoli G Theriot Corey A CA Hazra Tapas K TK Mitra Sankar S Lu A-Lien AL

Nucleic acids research 20070329 8

The checkpoint protein Rad9/Rad1/Hus1 heterotrimer (the 9-1-1 complex) is structurally similar to the proliferating cell nuclear antigen sliding clamp and has been proposed to sense DNA damage that leads to cell cycle arrest or apoptosis. Human (h) NEIL1 DNA glycosylase, an ortholog of bacterial Nei/Fpg, is involved in repairing oxidatively damaged DNA bases. In this study, we show that hNEIL1 interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. Residues 290-350 of hNEI ...[more]

PMID: 17395641

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The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase.

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The Rad9-Rad1-Hus1 DNA Repair Clamp is Found in Microsporidia.

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Identification of a candidate rad1 subunit for the kinetoplastid 9-1-1 (rad9-hus1-rad1) complex.

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DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex.

Project description:The Rad9-Rad1-Hus1 checkpoint clamp activates the DNA damage response and promotes DNA repair. DNA loading on the central channel of the Rad9-Rad1-Hus1 complex is required to execute its biological functions. Because Rad9A has the highest DNA affinity among the three subunits, we determined the domains and functional residues of human Rad9A that are critical for DNA interaction. The N-terminal globular domain (residues 1-133) had 3.7-fold better DNA binding affinity than the C-terminal globular domain (residues 134-266) of Rad9A1-266. Rad9A1-266 binds DNA 16-, 60-, and 30-fold better than Rad9A1-133, Rad9A134-266, and Rad9A94-266, respectively, indicating that different regions cooperatively contribute to DNA binding. We show that basic residues including K11, K15, R22, K78, K220, and R223 are important for DNA binding. The reductions on DNA binding of Ala substituted mutants of these basic residues show synergistic effect and are dependent on their residential Rad9A deletion constructs. Interestingly, deletion of a loop (residues 160-163) of Rad9A94-266 weakens DNA binding activity by 4.1-fold as compared to wild-type (WT) Rad9A94-266. Cellular sensitivity to genotoxin of rad9A knockout cells is restored by expressing WT-Rad9Afull. However, rad9A knockout cells expressing Rad9A mutants defective in DNA binding are more sensitive to H2O2 as compared to cells expressing WT-Rad9Afull. Only the rad9A knockout cells expressing loop-deleted Rad9A mutant are more sensitive to hydroxyurea than cells expressing WT-Rad9A. In addition, Rad9A-DNA interaction is required for DNA damage signaling activation. Our results indicate that DNA association by Rad9A is critical for maintaining cell viability and checkpoint activation under stress.

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