Streptococcus pneumoniae DNA polymerase I lacks 3''-to-5'' exonuclease activity: localization of the 5''-to-3'' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.     

Sequence and analysis of the gene for bacteriophage T3 RNA polymerase.

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.     

Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins.

The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.     

Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.     

Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.     

Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase

Bacteriophage T7 DNA polymerase, the product of gene 5 of the phage, has both polymerase and single-and double-stranded DNA 3'-to 5'-exonuclease activities. The exonuclease activities can be inactivated selectively by an oxidation reaction that requires molecular oxygen, a reducing agent, and iron at a concentration less than or equimolar to that of the gene 5 protein. Both exonuclease activities can be diminished by several thousandfold, with only a small decline in the polymerase activity. Escherichia coli thioredoxin, an accessory protein that binds tightly to the gene 5 protein and increases the processivity of the polymerization reaction, has no effect on the rate of oxidation. We propose that iron binds specifically to the exonuclease domain and, in the presence of molecular oxygen and a reducing agent, generates reactive oxygen species that selectively modify amino acid residues essential for the exonuclease activities.     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.     

The putative single-stranded DNA-binding protein of the filamentous bacteriophage, Ifl. Amino acid sequence of the protein and structure of the gene.

The protein product corresponding to the gene located in the region of the coliphage Ifl genome shown to contain the code for the single-stranded DNA (ssDNA)-binding proteins of all filamentous phages so far studied has been isolated from infected bacterial cells and its amino acid sequence determined. The mature protein contains 95 amino acids (calculated molecular mass 10553 Da). Its sequence corresponds to that predicted from the DNA sequence but lacks the initiating methionine residue. Although there is little direct sequence homology between the phage Ifl protein and the ssDNA-binding proteins of the other filamentous phages that have been studied, computer-based comparisons of various physical and structural parameters showed that the phage Ifl protein contains a domain that is closely related to domains in the coliphage T4 gene 32 protein and the Pseudomonas phage Pfl ssDNA-binding protein and suggest that the Ifl protein does have a ssDNA-binding function although we were unable to show this directly.     

The complete nucleotide sequence of the left very early region of Escherichia coli bacteriophage PRD1 coding for the terminal protein and the DNA polymerase

DNA molecules replicating in a linear form have been found in certain viruses and plasmids of both prokaryotic and eukaryotic origin. Characteristic of this type of molecules are the proteins covalently linked to their 5' ends and inverted terminal nucleotide sequences. The molecules replicate via a protein-priming mechanism, where participants include terminal protein and a specific polymerase. We report here the nucleotide sequence of the left very early region of Escherichia coli bacteriophage PRD1. This region codes for the terminal protein and the phage DNA polymerase. The predicted amino acid sequence of the terminal protein does not share homology with those of other known terminal proteins. The PRD1 DNA polymerase shows four regions of extensive homology to that of Bacillus subtilis phage phi 29. One of these conserved regions is also found in several animal virus DNA polymerases.     

Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.     

The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase.

T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.     

Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.     

Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene

A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.     

Mapping of the DNA linking tyrosine residue of the PRD1 terminal protein.

DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5' terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (phi 29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (phi 29) and serine-577 (adenovirus-2) are found in beta-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a beta-sheet in a hydrophobic region.     

Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5

We have determined the nucleotide sequence of a region of adenovirus type 5 (Ad5) DNA located between map positions 61.7 and 71.4, which covers the gene form the 72 kD DNA binding protein (DBP) and the sequence encoding the amino-terminal part of the 100 kD protein. Sequence analysis of cDNA copies of DBP mRNA revealed the existence of two abundant species of spliced mRNA molecules. One species consists of two short leader sequences from positions 75.2 (67 and 68 nucleotides long) and 68.8 (77 nucleotides long), respectively, and the main body of the RNA molecules. The other species contains only the leader sequence from position 75.2 and the main body. The amino acid sequence of DBP is encoded entirely by a long open reading frame of 1587 nucleotides in the main body of DBP mRNA. From the nucleotide sequence of the DBP gene it can be derived that DBP contains 529 amino acid residues and has an actual molecular weight of 59,049 daltons. The sites of mutation in the mutants H5hr404 and H5ts125 were determined at the nucleotide level. Single nucleotide alterations were detected in H5hr404 and H5ts125 in the sequences corresponding to the amino-terminal part and the carboxy-terminal part of DBP, respectively. The implications of these mutations are discussed.     

Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family.

A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name "EX1HH-HX3H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The "His" motifs often alternate with amino- and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29-54[CH]X2,3[CH]. The EX1HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.     

Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions.

epsilon BP (for epsilon binding protein) is a M(r) 31,000 S-type animal lectin that binds to IgE and has been identified as the homologue of Mac-2, a macrophage cell-surface marker, as well as the lectins RL-29, CBP35, and L-34. The protein is composed of two domains with the amino-terminal portion containing tandem repeats of nine amino acids and the carboxyl-terminal half containing consensus sequences shared by S-type animal lectins. We determined the genomic map in both rat and mouse and isolated overlapping genomic clones that contain the 5' two-thirds of the murine gene. The remaining portion of the gene was obtained by polymerase chain reaction (PCR) amplification of genomic murine DNA followed by subcloning into plasmid vectors. The epsilon BP gene is composed of six exons separated by five introns. The entire amino-terminal repetitive sequence is contained in exon III, and the carboxyl-terminal domain is encoded by the three succeeding exons (IV, V, VI). The latter three exons correspond well in size and share sequence homology with three exons coding for 14-kDa S-type lectins. The sequence in exon I offers an explanation for the generation of two mRNAs differing only in their 5' untranslated sequences, previously reported in Mac-2 cDNA clones. Using cDNA synthesis and PCR amplification, we determined that two alternative splice sites are used in many different types of cells. This alternative splicing results in different 5' untranslated regions of the murine epsilon BP mRNA.