TY - JOUR
T1 - Evidence for an imino intermediate in the DNA polymerase β deoxyribose phosphate excision reaction
AU - Piersen, Colleen E.
AU - Prasad, Rajendra
AU - Wilson, Samuel H.
AU - Lloyd, R. Stephen
PY - 1996
Y1 - 1996
N2 - A recent study demonstrated that rat DNA polymerase β (β-pol) releases 5'-deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during certain types of base excision repair. This catalytic activity resides within the amino-terminal, 8-kDa domain of β-pol and occurs via β-elimination as opposed to hydrolysis (Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702). The latter finding suggested that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap β-pol on preincised apurinic/apyrimidinic DNA using NaBH4 as the reducing agent. Both 8-kDa domain-DNA and intact β-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting. Our results indicate that the chemical mechanism of the β-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.
AB - A recent study demonstrated that rat DNA polymerase β (β-pol) releases 5'-deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during certain types of base excision repair. This catalytic activity resides within the amino-terminal, 8-kDa domain of β-pol and occurs via β-elimination as opposed to hydrolysis (Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702). The latter finding suggested that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap β-pol on preincised apurinic/apyrimidinic DNA using NaBH4 as the reducing agent. Both 8-kDa domain-DNA and intact β-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting. Our results indicate that the chemical mechanism of the β-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.
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U2 - 10.1074/jbc.271.30.17811
DO - 10.1074/jbc.271.30.17811
M3 - Article
C2 - 8663612
AN - SCOPUS:0029892846
SN - 0021-9258
VL - 271
SP - 17811
EP - 17815
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 30
ER -