The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase.
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.
Guan
Xin
X
Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
Madabushi
Amrita
A
Chang
Dau-Yin
DY
Fitzgerald
Megan E
ME
Shi
Gouli
G
Drohat
Alexander C
AC
Lu
A-Lien
AL
D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004789:Enzyme Activation; D005092:Exonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D044502:Thymine DNA Glycosylase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; C549605:HUS1 protein, human; C462949:HUS1B protein, human; C069930:rad9 protein; D005092:Exonucleases; C113574:Rad1 protein, human; D044502:Thymine DNA Glycosylase