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

Abstract

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.

Genome Protection by the 9-1-1 Complex Subunit HUS1 Requires Clamp Formation, DNA Contacts, and ATR Signaling-independent Effector Functions

“…That cells expressing these pocket mutants were not as hypersensitive to genotoxic stress as cells that completely lack HUS1 probably relates to the fact that they remain functional for genotoxin-induced CHK1 activation, as would be expected because interactions between 9-1-1 and the ATR activator TOPBP1 occur through the C-terminal tail of RAD9A (17). It also remains possible that RAD9A and RAD1 provide some level of redundancy when HUS1 is dysfunctional, because in some cases, RAD9A, HUS1, and RAD1 can all interact with the same repair protein, albeit with different binding affinities (42)(43)(44)(45)(46)(47). We favor the possibility that each 9-1-1 subunit binds at least some unique downstream effectors, with the idea that each subunit is specialized to some extent to mediate specific functions.…”

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

“…In contrast to PCNA, the 9-1-1 complex is thought to serve as a recruitment platform to bring checkpoint effector kinases to sites of DNA damage, thus activating checkpoint control, and also functions to stabilize stalled replication forks that have encountered DNA lesions (21)(22)(23). It has also been demonstrated that 9-1-1 interacts with and stimulates enzymes involved in base excision repair (BER), such as NEIL1, MYH, TDG, FEN1, and DNA Ligase I, thus potentially linking BER activities to checkpoint coordination (24)(25)(26)(27).…”

Crystal structure of human thymine DNA glycosylase bound to DNA elucidates sequence-specific mismatch recognition

“…4). Although it has been suggested that the very slow catalytic turnover of hTDG (7,8,12) is attributable largely to nonspecific DNA interactions involving the N-terminal Ϸ120 residues (13), the catalytic core (hTDG cat ) is also strongly product inhibited (6,14,37). The extensive interactions observed here with the abasic nucleotide and backbone phosphates of both DNA strands, including the R275 side-chain contacts, offer a structural explanation for the slow turnover of hTDG cat and hTDG.…”

“…Absent a clear role in catalysis, it is conceivable that the 2:1 complex, if adopted in vivo, could be important for some other function of hTDG. The enzyme is known to interact with proteins involved in transcriptional regulation, with the Dnmt3a/b methyltransferases and the Rad9-Rad1-Hus1 cell cycle checkpoint complex (6,14,28). Additional studies are needed to fully explore conditions that favor 2:1 binding and its potential biological role(s).…”

“…These enzymes excise a variety of damaged bases (X), and typically exhibit a strong preference for lesions in G⅐X versus A⅐X pairs (7-12). Like its eukaryotic orthologs, hTDG (410 residues) contains a conserved catalytic core (residues 121-300) flanked by more divergent Nand C-terminal domains (6); the former enhances DNA binding and G⅐T repair activity to some extent (13,14), and the latter contains a site for SUMO conjugation (K330), a modification that decreases the DNA-binding affinity of hTDG (15,16).A recent structure of the hTDG catalytic domain (residues 117-332, conjugated to SUMO-1) reveals strong similarity to the structure of eMUG (16,17), consistent with the 32% amino acid sequence identity. Nevertheless, the specificity of these enzymes differs remarkably.…”