DNA integrity is essential to life, however DNA is constantly subject to assaults from endogenous and exogenous species leading to the production of various types of DNA lesions. Many repair pathways are employed to find and repair these lesions. Of these pathways the base-excision repair pathway (BER) deals with the majority of DNA lesions. This repair is initiated by DNA glycosylases that recognize a base mismatch in DNA and remove that base. We are particularly interested in how DNA glycosylases recognize and catalyze their substrate nucleobase lesions in millions-fold excess of normal bases. One protein of interests is methyl-CpG-binding domain protein 4 (MBD4) which is known to bind the methylated DNA of the CpG repeating regions, and also to remove T from T:G mismatched DNA. Here we present three structures of catalytically relevant T:G and T:O6MeG mismatch intermediates of MBD4-DNA complexes and Arg468Lys MBD4-DNA complex. The T:O6MeG mismatch intermediates of the MBD4-DNA complex showed a strong preference for thymine binding in the flipped-out base binding site despite of the G to O6MeG mutation. We found that Arg468 residue of MBD4 is critical to stabilize the unpaired DNA primer base and the adjacent phosphate backbone of the flipped-out base of the DNA template thymine. The Arg468Lys MBD4 shows similar properties to Arg468 MBD4 in structure and enzyme activity in contrast to other Arg468 mutants which lost their activities.

Structural basis for mismatch recognition and catalysis by methyl-CpG-binding domain protein 4

247th National Meeting (2014)

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