Kinetic basis of nucleotide selection employed by a protein template-dependent DNA polymerase.
Rev1, a Y-family DNA polymerase, contributes to spontaneous and DNA damage-induced mutagenic events. In this paper, we have employed pre-steady-state kinetic methodology to establish a kinetic basis for nucleotide selection by human Rev1, a unique nucleotidyl transferase that uses a protein template-directed mechanism to preferentially instruct dCTP incorporation. This work demonstrated that the high incorporation efficiency of dCTP is dependent on both substrates: an incoming dCTP and a templating base dG. The extremely low base substitution fidelity of human Rev1 (10(0) to 10(-5)) was due to the preferred misincorporation of dCTP with templating bases dA, dT, and dC over correct dNTPs. Using non-natural nucleotide analogues, we showed that hydrogen bonding interactions between residue R357 of human Rev1 and an incoming dNTP are not essential for DNA synthesis. Lastly, human Rev1 discriminates between ribonucleotides and deoxyribonucleotides mainly by reducing the rate of incorporation, and the sugar selectivity of human Rev1 is sensitive to both the size and orientation of the 2'-substituent of a ribonucleotide.
Brown
Jessica A
JA
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Fowler
Jason D
JD
Suo
Zucai
Z
D004261:DNA Replication / Q000235:genetics*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003849:Deoxyguanosine / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics*; D013379:Substrate Specificity; D013698:Templates, Genetic; D000099270:Y-Family DNA Polymerases
D003845:Deoxycytosine Nucleotides; D003849:Deoxyguanosine; D009687:Nuclear Proteins; D009711:Nucleotides; D009713:Nucleotidyltransferases; D011506:Proteins; C024107:2'-deoxycytidine 5'-triphosphate; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases