TY - JOUR
T1 - Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase
AU - Adhikari, Sanjay
AU - Üren, Aykut
AU - Roy, Rabindra
N1 - Funding Information:
We thank the Biacore Molecular Interaction Shared Resource of the Lombardi Comprehensive Cancer Center for SPR experiments supported by P30 CA51008. We thank Ms. Karen Howenstein for expert editorial service. We thank Dr. Partha S. Mitra of Georgetown University for critically reading the paper. The work was supported by NIH grants RO1 CA 92306 (RR) and RO1 CA 108641 (AU).
PY - 2009/10/2
Y1 - 2009/10/2
N2 - 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 excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N6-ethenoadenine (εA) from εA-containing oligonucleotide (εA-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (KD) towards MPG, but under single-turnover (STO) condition there is apparently no effect on catalytic chemistry; however, the turnover of the enzyme under multiple-turnover (MTO) condition is higher for εA-DNA than it is for Hx-DNA. Real-time binding experiments by SPR spectroscopy further showed that the dissociation of MPG from its product, AP-site containing DNA, is faster than the overall turnover of either Hx- or εA-DNA reaction. We thereby conclude that the excised base plays a critical role in product inhibition and, hence, is essential for MPG glycosylase activity. Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions.
AB - 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 excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N6-ethenoadenine (εA) from εA-containing oligonucleotide (εA-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (KD) towards MPG, but under single-turnover (STO) condition there is apparently no effect on catalytic chemistry; however, the turnover of the enzyme under multiple-turnover (MTO) condition is higher for εA-DNA than it is for Hx-DNA. Real-time binding experiments by SPR spectroscopy further showed that the dissociation of MPG from its product, AP-site containing DNA, is faster than the overall turnover of either Hx- or εA-DNA reaction. We thereby conclude that the excised base plays a critical role in product inhibition and, hence, is essential for MPG glycosylase activity. Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions.
KW - Base excision repair
KW - Glycosylase
KW - N-Methylpurine-DNA glycosylase (MPG)
KW - Product inhibition
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U2 - 10.1016/j.dnarep.2009.06.005
DO - 10.1016/j.dnarep.2009.06.005
M3 - Article
C2 - 19616486
AN - SCOPUS:70149108748
SN - 1568-7864
VL - 8
SP - 1201
EP - 1206
JO - DNA Repair
JF - DNA Repair
IS - 10
ER -