Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage.

Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.

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

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.

Synthesis of polyribonucleotide chains from the 3'-hydroxyl terminus of oligodeoxynucleotides by Escherichia coli primase.

Escherichia coli primase synthesizes RNA primers on DNA templates for the initiation of DNA replication. The sole known activity of primase is to catalyze synthesis of short RNA chains de novo. We now report a novel activity of primase, namely that it can synthesize RNA from the 3'-hydroxyl terminus of a pre-existing oligodeoxynucleotide. The oligonucleotide-primed synthesis of RNA by primase occurs in both of the G4oric-specific priming system and the dnaB protein associated general priming system. This priming reaction of primase is verified by a number of biochemical methods, including inhibition by modified 3'-phosphate of oligonucleotides and deoxyribonuclease I and ribonuclease H cleavages. We also show that the primed RNA is an effective primer for the synthesis of DNA chain by E. coli DNA polymerase III holoenzyme. The significance of this finding to primases generating multimeric length RNA is discussed.

Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis.

Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication. By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4oric fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein. Together with the previous data, we have defined the structure of the primase/SSB/G4oric priming complex. Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 3' and 5' bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure. Two primase molecules then bind separately to specific DNA sequences in the 3' and 5' SSB-free G4oric regions. Binding of the 3' SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming. The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems. Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template.

ATP cross-linked to Escherichia coli single-strand DNA-binding protein can be utilized by the catalytic center of primase as initiating nucleotide for primer RNA synthesis on phage G4oric template.

We report a new observation of the role of Escherichia coli single-strand DNA binding protein (SSB) in synthesis of primer RNA (pRNA) catalyzed by.E.coli primase on the SSB-coated phage G4oric template. Using a set of ATP priming substrates with reactive groups attached to the 5' gamma-phosphate on different length "arms", we have demonstrated that, in the primase/SSB/G4oric pRNA synthesis complex, ATP cross-linked to both primase and SSB could be equally utilized as initiating nucleotide for pRNA synthesis. The distance between SSB surface and alpha-phosphorus of the priming substrate was estimated to be less than 7 A. ATP cross-linked to primase and SSB can be further elongated in the presence of other NTPs, giving almost identical patterns of covalently attached pRNAs of up to 12 nucleotides in length. The regions of primase and SSB with cross-linked ATP that can be used for pRNA synthesis are, therefore, arranged in a similar way relative to the active center of pRNA synthesis. The pRNA covalently linked to SSB was localized, mapping between Met48 and Trp88. This observation raises the possibility that SSB may play an active role in the initiation of pRNA synthesis in this system.

Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex.

We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori(c)), leaving stem-loop I and the adjacent 5' CTG 3', the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson, J. Biol. Chem. 268:8026-8039, 1993). Using a small 278-nucleotide (nt) G4ori(c) single-stranded DNA fragment that supported primer RNA synthesis, we now demonstrate by gel shift that E. coli primase can stably interact with the SSB-G4ori(c) complex. This stable interaction requires Mg2+ for specificity. At 8 mM Mg2+, primase binds to an SSB-coated 278-nt G4ori(c) fragment but not to an SSB-coated control 285-nt LacZ ss-DNA fragment. In the absence of Mg2+, primase binds to both SSB-coated fragments and gives a gel shift. T4 gene 32 protein cannot substitute for E. coli SSB in this reaction. Stable interaction of primase with naked G4ori(c). single-stranded DNA was not observed. DNase I and micrococcal nuclease footprinting, of both 5' and 3' 32P-labeled DNA, demonstrated that primase interacts with two regions of G4ori(c): one covering stem-loop I and the 3' sequence flanking stem-loop I which contains the pRNA initiation site and another located on the 5' sequence flanking stem-loop III.

Studies of the functional topography of the catalytic center of Escherichia coli primase.

The catalytic center of E. coli primase (581 amino acids) was identified by using, in the G4oric single-strand binding protein (SSB) primer RNA (pRNA) synthesis system, ATP and AMP derivatives, which were modified on the 5' side with reactive groups that can be cross-linked to the ATP binding site plus [alpha-32P]GTP. The position of the covalently attached 32P-labeled dinucleotide was mapped by chemical and enzymatic cleavage of labeled wild type and deletion mutants of primase. The catalytic center involves one of the Lys residues Lys-211, Lys-229, and Lys-241. The ATP binding site is preformed in primase, and the cross-linked ATP residue can be elongated to a 5-nucleotide limit, which implies significant stretching of the catalytic center during pRNA synthesis. His-43 close to the N terminus in a proposed zinc finger and Lys-528 near the C terminus were also cross-linked to ATP residues in the primase ATP binding site, suggesting that these regions are topographically close to the catalytic center during pRNA synthesis. When cross-linking was performed on the preformed primase/SSB/G4oric complex with long arm reagents (12-15 A), SSB was also labeled, indicating a close proximity to the site of pRNA synthesis.

Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454.

By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single-stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate.

Domains of Escherichia coli primase: functional activity of a 47-kDa N-terminal proteolytic fragment.

Endoproteinase Asp-N cleaves the 581-amino acid Escherichia coli primase (65,564 Da) into several major fragments. One of these, a 47-kDa fragment containing the complete N terminus and the first 422 amino acids of primase, is capable of primer RNA (pRNA) synthesis in the G4oric/single-stranded DNA binding protein/primase pRNA synthesis system. A cloned 398-amino acid N-terminal fragment of primase can also synthesize pRNA. The sizes of the pRNA synthesized by these N-terminal fragments, however, are smaller than those synthesized by intact primase, suggesting that the C-terminal region of primase plays a role in processivity or regulation of pRNA synthesis. Primase mutants with the last 10 and 40 C-terminal amino acids deleted synthesize pRNA as wild-type primase, indicating that any regulatory sequences must be internal to the C terminus of primase.

Selective decay of Escherichia coli dnaG messenger RNA is initiated by RNase E.

The rpsU-dnaG-rpoD operon messenger RNA that encodes S21, primase, and sigma-70 is cleaved under normal physiological conditions. The dnaG coding portion of the mRNA is then rapidly degraded. An endonuclease activity has been isolated from wild type Escherichia coli cells that cleaves dnaG mRNA. This activity has been identified as RNase E, and the identity confirmed by the accumulation of the unprocessed operon polycistronic mRNA in RNase E mutants, rne-3071 and ams-1, when incubated at nonpermissive temperatures. Extracts prepared from RNase E mutant strains failed to cleave dnaG mRNA in vitro. The dnaG mRNA RNase E cleavage site (5'-AAGUGAUUUA-3') is only 50% homologous to the ribosomal RNA RNase E cleavage site. By using computer programs predicting secondary structure, the dnaG RNase E cleavage site appears to be in a single stranded RNA loop.

Binding and phasing of Escherichia coli single-stranded DNA-binding protein by the secondary structure of phage G4 origin of complementary DNA strand synthesis (G4oric).

The origin of phage G4 DNA complementary strand synthesis (G4oric) consists of three stem-loop structures (stem loops I, II, and III) that have been proposed as a recognition site for primase during primer RNA (pRNA) synthesis (Godson, G. N., Barrell, B. G., Staden, R., and Fiddes, J. C. (1978) Nat. New Biol. 276, 236-247; Fiddes, J. C., Barrell, B. G., and Godson, G. N. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1081-1085; Sims, J., Capon, D., and Dressler, D. (1979) J. Biol. Chem. 254, 12615-12628). It is generally considered that the double-stranded DNA stem-loop structure is not coated with Escherichia coli single-stranded DNA-binding protein (SSB), but is recognized by primase as naked DNA (Kornberg, A., and Baker, J. (1992) DNA Replication, 2nd Ed., p. 280, W. H. Freeman & Co., New York). Using small G4oric single-stranded DNA fragments of various sizes (302, 278, 149, and 100 nucleotides) consisting of the core 100-nucleotide stem-loop region plus differing lengths of 3'- and 5'-flanking sequence as substrates for gel retardation and DNase I and micrococcal nuclease digestion, we show that under conditions of pRNA synthesis, two SSB tetramers bind to the stem-loop structure. With increasing lengths of 5'- and 3'-flanking sequence, more SSB tetramers are added. Regardless of the number of SSB tetramers bound, however, the region of DNA containing the pRNA initiation site is always left accessible to nuclease digestion. In situ copper-phenanthroline footprinting of individual gel shift assembly intermediates shows that on the 302-nucleotide G4oric, the first two SSB tetramers assemble at random, but the addition of more SSB tetramers results in formation of a unique structure. In this structure, SSB tetramers protect both sides of stem loop III plus the intervening region between stem loops III and I, but leave most of stem loop I and the CTG pRNA initiation site accessible to copper-phenanthroline. Primase can only synthesize pRNA when the stem-loop structure is saturated with SSB and presumably in the unique configuration. The G4oric stem-loop structure therefore appears to dictate the phasing of SSB to leave a primase recognition site as free DNA.

An over-expression plasmid for Escherichia coli primase.

To facilitate the overexpression of Escherichia coli primase, the dnaG gene has been reconstructed using polymerase chain reaction to remove the 5' transcription terminator and the 3' RNA processing site. This construct was cloned into the T7 polymerase-transcribed expression vector, pET-3d. Cells containing the resulting plasmid (pGNG1) express up to 30% of the cellular protein as primase. The pGNG1-encoded primase has normal activity in synthesizing primer RNA on a single-stranded DNA template in vitro. Plasmid pGNG1 can also be used to synthesize [35S]methionine-labelled primase in in vitro transcription-translation systems. In addition, the small amount of transcription in the absence of T7 polymerase is sufficient to complement temperature-sensitive and amber dnaG chromosomal mutations in vivo. Plasmid pGNG1 can therefore be used not only to overproduce wild-type primase, but to change and manipulate the primase structure in vivo and in vitro. These mutant proteins can be overproduced and used for structural and functional studies.

Cloning and nucleotide sequence of a chromosomally encoded tetracycline resistance determinant, tetA(M), from a pathogenic, methicillin-resistant strain of Staphylococcus aureus.

This report describes the cloning and sequencing of a chromosomally encoded tetracycline resistance determinant from a clinical isolate of methicillin-resistant Staphylococcus aureus. On the basis of the sequence, the gene is in the tet(M) class, and it was shown that the S. aureus tetA(M) gene is induced at the level of transcription.

Structural features of the priming signal recognized by primase: mutational analysis of the phage G4 origin of complementary DNA strand synthesis.

45 mutations (insertion, deletion and base substitution) of the G4 Goric were tested for their functional activity in M13 and R199 in vivo. The critical mutants were also assayed for their ability to synthesize pRNA in vitro using SSB and primase. The results demonstrate that the secondary structure and spacing of stem-loops I and III are essential for Goric activity and that the 5'-CTG-3' sequence flanking stem-loop I is essential for initiation of pRNA synthesis.

Hypersensitive mung bean nuclease cleavage sites in Plasmodium knowlesi DNA.

Nucleotide sequences of Plasmodium knowlesi DNA that are cleaved by mung bean nuclease (Mbn) at low enzyme concentration (0.2 units enzyme per micrograms DNA) are listed. They are tandemly repeated purine/pyrimidine (RpY) stretches of DNA with (ApT) dimers predominating. Most cut sites are within almost 100% RpY tracts. The enzyme cleaves at many points within the RpY stretch and usually hydrolyzes the 5'-ApT-3' linkage. These alternating RpY target sites are flanked by homopurine and homopyrimidine stretches. At least one Mbn target site lies next to an in vivo transcribed region.

Isolation of alpha- and beta-tubulin genes of Plasmodium falciparum using a single oligonucleotide probe.

An oligonucleotide probe (315) specific for the alpha- and beta-tubulin genes of Plasmodium falciparum was synthesized utilizing codon usage of P. falciparum determined from published gene sequences. By screening genomic and cDNA libraries with the oligonucleotide probe, alpha- and beta-tubulin clones were isolated. Positive clones were identified by partial sequencing and comparing the deduced amino acid sequence with the chicken brain alpha- and beta-tubulin amino acid sequences. The beta-tubulin gene was completely sequenced at the genomic level and partially at cDNA level. The deduced polypeptide is 445 amino acids long, shares 88% homology with chicken brain beta-tubulin, and contains two introns of 362 and 163 bp long, respectively. alpha- and beta-tubulin genes of P. falciparum are unlinked and dispersed; more than one copy of each gene may be present. Northern blot analysis of total RNA of the blood-stage parasite indicates the presence of three transcripts of alpha-tubulin (3.3 kb, 2.6 kb, 1.9 kb) and three transcripts of beta-tubulin gene (3.6 kb, 2.9 kb, 2.0 kb). The significance of these transcripts is presently unknown.

Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase.

The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity. However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function.

Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis.

Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I, and II, and/or II and III, might play a role in G4oric function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4oric activity. This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity. To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity.

DNA----DNA, and DNA----RNA----protein: orchestration by a single complex operon.

In Escherichia coli, the workhorse of molecular biology, a single operon is involved in the replication, transcription and translation of genetic information. This operon is controlled in a complex manner involving multiple cis-acting regulatory sequences and trans-acting regulatory proteins. It interacts with global regulatory networks by mechanisms which are presently being dissected.

An Escherichia coli cis-acting antiterminator sequence: the dnaG nut site.

The Escherichia coli rpsU-dnaG-rpoD operon contains an internal transcription terminator T1 located in the intergenic region between the rpsU and dnaG genes (Smiley et al. 1982). By cloning T1 as a small 127 bp fragment into the terminator probe plasmid pDR720 between the trp operator promoter and the assayable galK gene, it was shown that T1 acts as a strong transcription terminator, comparable in strength to the 3' operon terminator T2. However, an operon sequence that occurs 5' to T1 within the coding region of the rpsU gene and which has homology with the lambda nut site, (Lupski et al. 1983) when placed 5' to T1 in the pDR720 plasmid construct, modifies transcription through T1 allowing expression of the galK gene. This sequence, called the dnaG nut site also modifies the termination activity of the external operon terminator T2. It is proposed that the dnaG nut site is a cis-acting element of an antitermination system in E. coli.