Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: Primase and the family X polymerases share significant sequence homology

Comparison of the amino acid sequences of eucaryotic DNA primase and the fa mily X polymerases indicates that primase shares significant sequence homol ogy with this family. With the use of DNA polymerase beta (pol beta) as a p aradigm for family X polymerases, these homologies include both the catalyt ic core domain/subunit of each enzyme (31 kDa domain of pol beta and p49 su bunit of primase) as well as the accessory domain/subunit (8 kDa domain of pol beta and p58 subunit of primase). To further explore these homologies a s well as provide insights into the mechanism of primase, we generated thre e mutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved between primase and the eukaryotic family X polymerases . These mutations significantly decreased the rate of primer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlated with the severity of the mutation (A > Q > K). R304 also contrib utes to efficient utilization of the NTP that will become the 5'-terminus o f the new primer, and these effects are at least partially mediated through interactions with the phosphates of this NTP. The implications of these re sults with respect to the structure and biological role of primase, as well as its relationship to the family X polymerases, are discussed.