Nucleic Acids Research Advance Access originally published online on September 12, 2007
Nucleic Acids Research 2007 35(18):6207-6218; doi:10.1093/nar/gkm678
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Nucleic Acids Research, 2007, Vol. 35, No. 18 6207-6218
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase
Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, School of Medicine, University of Maryland, Baltimore, MD 21201, USA
*To whom correspondence should be addressed. Tel: +1 410 706 4356; Fax: +1 410 706 1787; Email: aluchang{at}umaryland.edu
Received May 18, 2007. Revised August 15, 2007. Accepted August 18, 2007.
Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9–Rad1–Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67–110 of hTDG, and Val74 of hTDG plays an important role in the TDG–Hus1 interaction. In contrast to the core domain of hTDG (residues 110–308), hTDG(67–308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER.
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