Arg304 of Human DNA Primase Is a Key Contributor to Catalysis and NTP Binding: Primase and the Family X Polymerases Share Significant Sequence Homology
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- 作者：Brian W. Kirk and Robert D. Kuchta
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：June 15, 1999
- 年：1999
- 卷：38
- 期：24
- 页码：7727 - 7736
- 全文大小：197K
- 年卷期：v.38,no.24(June 15, 1999)
- ISSN：1520-4995
文摘
Comparison of the amino acid sequences of eucaryotic DNA primase and the family Xpolymerases indicates that primase shares significant sequence homology with this family. With the useof DNA polymerase 
(pol ) as a paradigm for family X polymerases, these homologies include boththe catalytic core domain/subunit of each enzyme (31 kDa domain of pol and p49 subunit of primase)as well as the accessory domain/subunit (8 kDa domain of pol and p58 subunit of primase). To furtherexplore these homologies as well as provide insights into the mechanism of primase, we generated threemutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved betweenprimase and the eukaryotic family X polymerases. These mutations significantly decreased the rate ofprimer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlatedwith the severity of the mutation (A > Q > K). R304 also contributes to efficient utilization of the NTPthat will become the 5'-terminus of the new primer, and these effects are at least partially mediated throughinteractions with the phosphates of this NTP. The implications of these results with respect to the structureand biological role of primase, as well as its relationship to the family X polymerases, are discussed.
(pol ) as a paradigm for family X polymerases, these homologies include boththe catalytic core domain/subunit of each enzyme (31 kDa domain of pol and p49 subunit of primase)as well as the accessory domain/subunit (8 kDa domain of pol and p58 subunit of primase). To furtherexplore these homologies as well as provide insights into the mechanism of primase, we generated threemutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved betweenprimase and the eukaryotic family X polymerases. These mutations significantly decreased the rate ofprimer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlatedwith the severity of the mutation (A > Q > K). R304 also contributes to efficient utilization of the NTPthat will become the 5'-terminus of the new primer, and these effects are at least partially mediated throughinteractions with the phosphates of this NTP. The implications of these results with respect to the structureand biological role of primase, as well as its relationship to the family X polymerases, are discussed.