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 family X polymerases indicates that primase shares significant sequence homology with this family. With the use of DNA polymerase beta (pol beta) as a paradigm for family X polymerases, these homologies include both the catalytic core domain/subunit of each enzyme (31 kDa domain of pol beta and p49 subunit 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 as well as provide insights into the mechanism of primase, we generated three 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 contributes to efficient utilization of the NTP that will become the 5'-terminus of the new primer, and these effects are at least partially mediated through interactions with the phosphates of this NTP. The implications of these results with respect to the structure and biological role of primase, as well as its relationship to the family X polymerases, are discussed.
Kirk
B W
BW
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309, USA.
Kuchta
R D
RD
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D019951:DNA Polymerase beta / Q000737:chemistry; D019915:DNA Primase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013698:Templates, Genetic
D009711:Nucleotides; D010710:Phosphates; D011994:Recombinant Proteins; D001120:Arginine; D019915:DNA Primase; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase