N-terminal extension of N-methylpurine DNA glycosylase is required for turnover in hypoxanthine excision reaction.
N-Methylpurine DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study we tested the role of N-terminal extension on MPG hypoxanthine (Hx) cleavage activity. Our results showed that MPG lacking N-terminal extension excises hypoxanthine with significantly reduced efficiency, one-third of that exhibited by full-length MPG under similar conditions. Steady-state kinetics showed full-length MPG has higher V(max) and lower K(m) than NDelta100 MPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that truncation can substantially increase the equilibrium binding constant of MPG toward Hx, but under single-turnover conditions there is apparently no effect on catalytic chemistry; however, the truncation of the N-terminal tail affected the turnover of the enzyme significantly under multiple turnover conditions. Real time binding experiments by surface plasmon resonance spectroscopy further showed that NDelta100 MPG binds approximately six times more tightly toward its product apurinic/apyrimidinic site than the substrate, whereas full-length MPG similarly binds to both the substrate and the product. We thereby conclude that the N-terminal tail in MPG plays a critical role in overcoming the product inhibition, which is achieved by reducing the differences of MPG binding affinity toward Hx and apurinic/apyrimidinic sites and thus is essential for the Hx cleavage reaction of MPG. The results from this study also affirm the need for reinvestigation of full-length MPG for its enzymatic and structural properties, which are currently available mostly for the truncated protein.
Adhikari
Sanjay
S
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057, USA.
Uren
Aykut
A
Roy
Rabindra
R
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D045647:DNA Glycosylases / Q000737:chemistry*; D004260:DNA Repair; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance
D009841:Oligonucleotides; D011994:Recombinant Proteins; D019271:Hypoxanthine; D045647:DNA Glycosylases