TY - JOUR
T1 - Active Site Interactions Impact Phosphoryl Transfer during Replication of Damaged and Undamaged DNA by Escherichia coli DNA Polymerase i
AU - Prakasha Gowda, A. S.
AU - Spratt, Thomas E.
N1 - Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/11/20
Y1 - 2017/11/20
N2 - Replicative DNA polymerases are able to discriminate between very similar substrates with high accuracy. One mechanism by which E. coli DNA polymerase I checks for Watson-Crick geometry is through a hydrogen bonding fork between Arg668 and the incoming dNTP and the minor groove of the primer terminus. The importance of the Arg-fork was examined by disrupting it with either a guanine to 3-deazaguanine substitution at the primer terminus or the use of a carbocyclic deoxyribose analog of dUTP. Using thio-substituted dNTPs and differential quench techniques, we determined that when the Arg-fork was disrupted, the rate-limiting step changed from a conformational change to phosphodiester bond formation. This result indicates that Arg668 is involved in the phosphoryl transfer step. We examined the role of the Arg-fork in the replication of four DNA damaged templates, O6-methylguanine (O6-mG), 8-oxo-7,8-dihydroguanine (oxoG), O2-[4-(3-pyridyl)-4-oxobutyl]thymine (O2-POB-T), and N2-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-8,9,10-trihydroxybenzo[a]pyren-7-yl]-guanine (N2-BP-G). In general, the guanine to 3-deazaguanine substitution caused a decrease in kpol that was proportional to kpol over five orders of magnitude. The linear relationship indicates that the Arg668-fork helps catalyze phosphoryl transfer by the same mechanism with all the substrates. Exceptions to the linear relationship were the incorporations of dTTP opposite G, oxoG, and O6mG, which showed large decreases in kpol, similar to that exhibited by the Watson-Crick base pairs. It was proposed that the incorporation of dTTP opposite G, oxoG, and O6mG occurred via Watson-Crick-like structures.
AB - Replicative DNA polymerases are able to discriminate between very similar substrates with high accuracy. One mechanism by which E. coli DNA polymerase I checks for Watson-Crick geometry is through a hydrogen bonding fork between Arg668 and the incoming dNTP and the minor groove of the primer terminus. The importance of the Arg-fork was examined by disrupting it with either a guanine to 3-deazaguanine substitution at the primer terminus or the use of a carbocyclic deoxyribose analog of dUTP. Using thio-substituted dNTPs and differential quench techniques, we determined that when the Arg-fork was disrupted, the rate-limiting step changed from a conformational change to phosphodiester bond formation. This result indicates that Arg668 is involved in the phosphoryl transfer step. We examined the role of the Arg-fork in the replication of four DNA damaged templates, O6-methylguanine (O6-mG), 8-oxo-7,8-dihydroguanine (oxoG), O2-[4-(3-pyridyl)-4-oxobutyl]thymine (O2-POB-T), and N2-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-8,9,10-trihydroxybenzo[a]pyren-7-yl]-guanine (N2-BP-G). In general, the guanine to 3-deazaguanine substitution caused a decrease in kpol that was proportional to kpol over five orders of magnitude. The linear relationship indicates that the Arg668-fork helps catalyze phosphoryl transfer by the same mechanism with all the substrates. Exceptions to the linear relationship were the incorporations of dTTP opposite G, oxoG, and O6mG, which showed large decreases in kpol, similar to that exhibited by the Watson-Crick base pairs. It was proposed that the incorporation of dTTP opposite G, oxoG, and O6mG occurred via Watson-Crick-like structures.
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U2 - 10.1021/acs.chemrestox.7b00257
DO - 10.1021/acs.chemrestox.7b00257
M3 - Article
C2 - 29053918
AN - SCOPUS:85034580284
SN - 0893-228X
VL - 30
SP - 2033
EP - 2043
JO - Chemical research in toxicology
JF - Chemical research in toxicology
IS - 11
ER -