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

Abstract

Earlier we showed by affinity cross-linking of initiating substrates to Escherichia 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 and 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 catalytic center. Primase with a mutation of Arg to Lys at position 241 (defined as K241R-primase) is almost unable to synthesize primer RNA (pRNA) on the single-stranded DNA-binding protein (SSB)/R199G4oric template. However, it is able to synthesize a pppApG dimer plus trace amounts of 8- to 11-nucleotide (nt) pRNA transcribed from the 5' CTG 3' pRNA initiation site on phage 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 elongation. In the general priming system, the K241R-primase also can synthesize only the dimer and very small amounts of 11-nt pRNA. The results of gel retardation experiments suggested that this deficiency in pRNA chain elongation of the K241R mutant primase is unlikely to be caused by impairment of the DNA binding activity. The K241R mutant primase, however, can still prime DNA synthesis in vivo and in vitro.

FIG. 1

Structure of primase and conservation of amino acid sequence surrounding Lys211, Lys229, and Lys241. (A) Domain structure of primase (data taken from references and 25). (B) Conserved amino acid sequences and motifs at the catalytic center of primase. Amino acids that are conserved in all 16 sequenced prokaryote primases are in bold type; the data were taken from Szfranski et al. (24). Conserved motifs 3 to 6 are from Ilyana et al. (7), and the RNA polymerase (RNAP), and dnaG boxes are from Verlasalovic et al. (26). The Lys211, Lys229, and Lys241 residues which were changed to Arg residues by oligonucleotide mutagenesis are indicated with arrows.

FIG. 2

Synthesis of pRNA by P47-K211R, P47-K229R, and P47-K241R mutant proteins. pRNA was synthesized on an SSB-coated R199/G4oric DNA template, as described in Materials and Methods. (A) pRNA synthesized by P47 and the mutant P47 proteins. Care was taken to use exactly the same amount of protein in each reaction mixture and to load an exact aliquot onto the gel (20% polyacrylamide–7 M urea gel with an acrylamide-to-bisacrylamide ratio of 19:1). The leftmost gel is a film exposed overnight. (B) In a similar experiment, the pRNA products were separated on a 23% polyacrylamide gel (acrylamide-to-bisacrylamide ratio of 8:1) containing 7 M urea. The gels were autoradiographed frozen. The size markers were [γ-³²P]ATP-labeled 12- and 18-nt oligonucleotides (oligo). The longer species of pRNA were 11 to 22 nt long, and the smaller species were deduced from their migration relative to that of free [α-³²P]GTP (not shown) to be the dimer(pppApG), trimer (pppApGpU), and tetramer (pppApGpUpA) pRNA.

FIG. 3

Synthesis of pRNA by the mutant P47 proteins with dideoxynucleotides to arrest chain extension. P47 and P47-K241R proteins were incubated under the normal pRNA synthesis conditions, except that dideoxynucleotides replaced specific rNTPs in order to terminate chain extension at different locations on the template. (A) Template sequence showing where the pRNA chain synthesized on G4oric would be terminated by ddTTP and ddCTP. (B) pRNA synthesized by P47 and P47-K241R in the presence of all four NTPs (lanes 2 and 5), and when ddTTP (ddT) (lanes 3 and 6) and ddCTP (ddC) (lanes 4 and 7) were added to the incubation mixture. pRNA products were separated on a 23% polyacrylamide gel (acrylamide-to-bisacrylamide ratio of 8:1) containing 7 M urea. The gel was run so that the free [α-³²P]GTP remained in the gel. Two autoradiographic exposures (15 s and 30 min, respectively) were necessary to visualize both free [α-³²P]GTP and the dimer or trimer pRNA.

FIG. 4

pRNA synthesis by primase-K241R. A six-His tag was fused to the N terminus of primase by using the vector pET-21d, and HT-primase was prepared by affinity purification on Ni resin. In the same vector, Lys241 was changed to Arg (HT-primase-K241R) by site-directed mutagenesis. (A) Comparison of the activity of HT-primase with two independent preparations of wild-type primase that does not contain a His tag. In this experiment, [α-³²P]UTP was used to label the pRNA products. Care was taken to use identical amounts of primase in each reaction mixture and to load identical aliquots of the reaction mixture on the polyacrylamide gel. (B) pRNA synthesis by HT-primase and HT-primase-K241R was assayed under the standard conditions with [α-³²P]GTP (see Materials and Methods). In both experiments, the synthesis products were separated on a 20% polyacrylamide–7 M urea gel (20 by 40 cm). To visualize the dimer pRNA in panel B, most of the [α-³²P]GTP was run out of the leftmost gel and the bottom region of the frozen gel was autoradiographed for a shorter time, as shown in the rightmost gel.

FIG. 5

pRNA synthesis by HT-primase-K241R in the general priming system. The pRNA synthesis of HT-primase and mutant HT-primase-K241R was assayed on R199 ssDNA in the presence of DnaB helicase. The products were separated on an small 18% polyacrylamide gel containing 7 M urea.

FIG. 6

Gel retardation assay of G4oric DNA binding by HT-primase-K241R. The wild-type or mutant primase was incubated with ³²P-labeled G4oric 278-nt ssDNA fragment and SSB, and binding complexes were then analyzed on a natural 4% polyacrylamide gel as described in Materials and Methods. The gel was dried before autoradiography. Lane 1, G4oric ssDNA alone; lane 2, DNA plus SSB; lane 3, DNA, SSB, and primase (no His tag); lane 4, DNA, SSB, and HT-primase; lane 5, DNA, SSB, and HT-primase-K241R. The shifted band induced by primase binding is indicated with an arrowhead.

FIG. 7

In vivo complementation chromosomal temperature-sensitive dnaG by plasmid-expressed K241R mutant primase. E. coli KY1378 containing temperature-sensitive dnaG2903 gene was transformed with pGNG7 (wild-type primase), pGNG7-K241R (mutant primase), and the control pET-21d vector (no insert). The transformed cells were plated on LB plates containing 100 μg of ampicillin per ml and incubated at 30 or 42°C overnight.

FIG. 8

In vitro DNA synthesis by Pol III holoenzyme with pRNA synthesized by HT-primase-K241R on 278-nt G4oric template. The synthesis reaction was performed in two stages; first pRNA synthesis and then DNA synthesis. [α-³²P]GTP was used to label the pRNA, and α-³⁵S-dATP was used to label the DNA, as described in Materials and Methods. The synthesis products were analyzed on an 8% polyacrylamide–7 M urea gel, which was dried before autoradiography. Lane 1, size markers of 5′-end γ-³²P-labeled fragments of pBR322 digested with MspI; lane 2, extension with wild-type HT-primase; lane 3, extension with mutant HT-primase-K241R; lane 4, extension with wild-type P47; lane 5, extension with mutant P47-K241R; lane 6, control of DNA synthesis by Pol III without adding primase in the first stage of the reaction.