A Conserved Tyr Residue Is Required for Sugar Selectivity in a Pol DNA Polymerase
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- 作者：Guangwei Yang ; Matthew Franklin ; Jing Li ; T.-C. Lin ; and William Konigsberg
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：August 13, 2002
- 年：2002
- 卷：41
- 期：32
- 页码：10256 - 10261
- 全文大小：113K
- 年卷期：v.41,no.32(August 13, 2002)
- ISSN：1520-4995
文摘
Many DNA polymerases select their natural substrates, deoxy- as opposed to ribonucleosidetriphosphates, with a selectivity greater than 10 000-fold. The function of a highly conserved residue,Tyr416, in the palm domain of the parental enzyme, an exo- derivative of RB69 DNA polymerase (gp43),a member of the pol 
chars/alpha.gif" BORDER=0> DNA polymerase family, was examined for its role in helping the polymerasediscriminate between ribo-, dideoxyribo-, and deoxyribonucleoside triphosphates. The parental enzymeselected dNTPs vs rNTPs with about the same preference as dNTPs vs ddNTPs. Pre-steady-state kineticanalysis was carried out with the parental enzyme and two mutants, Y416A and Y416F. The Y416Amutant incorporated ribonucleotide residues much more efficiently than the parental enzyme, whereas theY416F mutant was more permissive toward ddNTP vs rNTP utilization than either the Y416A mutant orthe parental enzyme. We also found that both dCDP and rCDP inhibited dCTP incorporation by theY416A mutant, while only dCDP but not rCDP inhibited dCTP incorporation by the parental enzyme andthe Y416F mutant. The parental enzyme and the Y416A and Y416F mutants were all able to add araCTP(1-chars/beta2.gif" BORDER=0 ALIGN="middle">-D-arabinofuranosylcytosine-5'-triphosphate) to a primer but with reduced efficiency relative to dCTP.Based on our kinetic results, interpreted in the context of the crystal structure of the RB69 gp43 ternarycomplex, we suggest that sugar discrimination is provided mainly by the Tyr416 side chain which cansterically block the 2'-OH group of an incoming rNTP.
chars/alpha.gif" BORDER=0> DNA polymerase family, was examined for its role in helping the polymerasediscriminate between ribo-, dideoxyribo-, and deoxyribonucleoside triphosphates. The parental enzymeselected dNTPs vs rNTPs with about the same preference as dNTPs vs ddNTPs. Pre-steady-state kineticanalysis was carried out with the parental enzyme and two mutants, Y416A and Y416F. The Y416Amutant incorporated ribonucleotide residues much more efficiently than the parental enzyme, whereas theY416F mutant was more permissive toward ddNTP vs rNTP utilization than either the Y416A mutant orthe parental enzyme. We also found that both dCDP and rCDP inhibited dCTP incorporation by theY416A mutant, while only dCDP but not rCDP inhibited dCTP incorporation by the parental enzyme andthe Y416F mutant. The parental enzyme and the Y416A and Y416F mutants were all able to add araCTP(1-chars/beta2.gif" BORDER=0 ALIGN="middle">-D-arabinofuranosylcytosine-5'-triphosphate) to a primer but with reduced efficiency relative to dCTP.Based on our kinetic results, interpreted in the context of the crystal structure of the RB69 gp43 ternarycomplex, we suggest that sugar discrimination is provided mainly by the Tyr416 side chain which cansterically block the 2'-OH group of an incoming rNTP.