A mutant Escherichia coli primase defective in elongation of primer RNA chains

Earlier we showed by affinity cross-linking of initiating substrates to Esc herichia coli primase that one or more of the residues Lys211, Lys229, and Lys241 were involved in the catalytic center of the enzyme (A, A. Mustaev a nd G. N. Godson, J. Biol, Chem, 270:15711-15718, 1995). We now demonstrate by mutagenesis that only Lys241 but not Lys211 and Lys229 is part of the ca talytic center. Primase with a mutation of Arg to Lys at position 241 (defi ned as K241R-primase) is almost unable to synthesize primer RNA (pRNA) on t he single-stranded DNA-binding protein (SSB)/R199G4oric template. However, it is able to synthesize a pppApG dimer plus trace amounts of 8- to Il-nucl eotide (nt) pRNA transcribed from the 5' CTG 3' pRNA initiation site on pha ge G4 oric DNA, The amount of dimer synthesized by K241R-primase is similar to that synthesized by the wild-type primase, demonstrating that the K241R mutant can initiate pRNA synthesis normally but is deficient in chain elon gation. In the general priming system, the K241R-primase also can synthesiz e only the dimer and very small amounts of Il-nt pRNA. The results of gel r etardation experiments suggested that this deficiency in pRNA chain elongat ion of the K241R mutant primase is unlikely to be caused by impairment of t he DNA binding activity. The K241R mutant primase, however, can still prime DNA synthesis in vivo and in vitro.