Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication

Hobbs, Lisa J.; Nossal, Nancy G.

doi:10.1128/jb.178.23.6772-6777.1996

Cited by 23 publications

(21 citation statements)

References 26 publications

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“…Although the nuclease can degrade from nicks when the gene 45 replication clamp is loaded behind it, this degradation is not processive and is much slower than that of the nuclease with 32 protein. 2 We conclude from these experiments that 32 protein controls the processing of Okazaki fragments. The presence of 32 protein guarantees that the nuclease will degrade processively, removing adjacent DNA as well as the pentamer RNA primers.…”

Section: Discussionmentioning

confidence: 73%

“…For bacteriophage T4 DNA replication, the primers are removed by a phage-encoded nuclease, T4 RNase H, that degrades both RNA⅐DNA and DNA⅐DNA duplexes from their 5Ј termini, giving short oligonucleotide products (1). T4 phage with a deletion in the RNase H gene have a reduced burst on a wild type host and cannot replicate in an Escherichia coli host defective in the 5Ј-to 3Ј-nuclease of pol I (2).…”

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confidence: 99%

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Bacteriophage T4 RNase H Removes Both RNA Primers and Adjacent DNA from the 5′ End of Lagging Strand Fragments

Bhagwat¹,

Nossal²

2001

Journal of Biological Chemistry

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Bacteriophage T4 RNase H belongs to a family of prokaryotic and eukaryotic nucleases that remove RNA primers from lagging strand fragments during DNA replication. Each enzyme has a flap endonuclease activity, cutting at or near the junction between single-and double-stranded DNA, and a 5-to 3-exonuclease, degrading both RNA⅐DNA and DNA⅐DNA duplexes. On model substrates for lagging strand synthesis, T4 RNase H functions as an exonuclease removing short oligonucleotides, rather than as an endonuclease removing longer flaps created by the advancing polymerase. The combined length of the DNA oligonucleotides released from each fragment ranges from 3 to 30 nucleotides, which corresponds to one round of processive degradation by T4 RNase H with 32 single-stranded DNA-binding protein present. Approximately 30 nucleotides are removed from each fragment during coupled leading and lagging strand synthesis with the complete T4 replication system. We conclude that the presence of 32 protein on the single-stranded DNA between lagging strand fragments guarantees that the nuclease will degrade processively, removing adjacent DNA as well as the RNA primers, and that the difference in the relative rates of synthesis and hydrolysis ensures that there is usually only a single round of degradation during each lagging strand cycle.During the replication of duplex DNA, the leading strand is synthesized continuously, but the lagging strand is synthesized as a series of fragments, each initiated by a short RNA primer made by a primase. Ultimately these RNA primers must be removed, the resulting gaps filled in by polymerase, and the adjacent fragments sealed together by DNA ligase. For bacteriophage T4 DNA replication, the primers are removed by a phage-encoded nuclease, T4 RNase H, that degrades both RNA⅐DNA and DNA⅐DNA duplexes from their 5Ј termini, giving short oligonucleotide products (1). T4 phage with a deletion in the RNase H gene have a reduced burst on a wild type host and cannot replicate in an Escherichia coli host defective in the 5Ј-to 3Ј-nuclease of pol I (2).T4 RNase H is a protein of 305 amino acids with significant sequence similarity to other prokaryotic and eukaryotic enzymes that remove RNA primers during DNA replication (3).These include the T7 gene 6 exonuclease, the N-terminal domain of E. coli pol I, Saccharomyces cerevisiae Rad27, and human FEN-1 proteins. These enzymes can degrade DNA⅐DNA as well as RNA⅐DNA duplexes. This raises the possibility that some DNA adjacent to the primers is removed and repaired in all of these replication systems. If there is reduced fidelity in the initial elongation of the primers, removing the adjacent DNA would improve replication accuracy.T4 RNase H by itself is a nonprocessive nuclease, removing a single short oligonucleotide (1-4 nucleotides) each time it binds to the substrate (4). In multiple turnover reactions, this degradation of the DNA duplex from the 5Ј end continues until a limit product of 8 -11 nucleotides remains at the 3Ј end. T4 gene 32 protein, binding on single...

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Section: Discussionmentioning

confidence: 73%

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confidence: 99%

Bacteriophage T4 RNase H Removes Both RNA Primers and Adjacent DNA from the 5′ End of Lagging Strand Fragments

Bhagwat¹,

Nossal²

2001

Journal of Biological Chemistry

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“…3). Replication is mediated by T4-encoded enzymes, with the exception that the host RNA polymerase synthesizes the primers for leadingstrand initiation at origins (577,668) and that host DNA polymerase I can remove RNA primers of Okazaki fragments (427). T4 primase then synthesizes primers for Okazaki fragments.…”

Section: Initiation Of Dna Replicationmentioning

confidence: 99%

Bacteriophage T4 Genome

Miller

Kutter

Mosig

et al. 2003

Microbiol Mol Biol Rev

686

801

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SUMMARY Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

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“…However, it also acts as a 5Ј-nuclease on DNA duplexes (3). Genetic studies indicate that either T4 RNase H or the 5Ј-to 3Ј-exonuclease of the Escherichia coli host DNA polymerase I is necessary for phage production (4). A T4 mutant with a large deletion (⌬118 -305) in the rnh gene gives a burst size of 50% of wild type T4 phage in a wild type host, but a burst of only a few phage per infected cell under nonpermissive conditions in E. coli PolA12, which has a conditionally lethal mutation in the host nuclease.…”

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Maturation of Bacteriophage T4 Lagging Strand Fragments Depends on Interaction of T4 RNase H with T4 32 Protein Rather than the T4 Gene 45 Clamp

Gangisetty¹,

Jones

Bhagwat³

et al. 2005

Journal of Biological Chemistry

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In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278 -305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.DNA synthesis on the lagging strand of the replication fork is accomplished by the rapid repetition of a cycle in which primase makes a short RNA primer that is elongated by polymerase; the primer is removed from the previous fragment, and the two fragments are joined by DNA ligase. The efficient sealing of adjacent fragments is essential to maintain the accuracy of DNA replication. The accumulation of nicks and gaps on the lagging strand ultimately results in double-stranded breaks, increased mutation frequencies, and cell lethality (reviewed in Refs. 1 and 2). The lagging strand cycle must be repeated every few seconds because the discontinuous fragments are so short, 1-2 kb in prokaryotes and less than 200 bases in eukaryotes. In each of the replication systems studied, the primers are removed by a member of a family of 5Ј-nucleases with conserved sequences and similar structures (Fig. 1). It is important that the process of primer removal from one fragment is coordinated with the elongation of the next fragment to ensure rapid and accurate replication. Our studies indicate that the mechanism by which lagging strand polymerization and primer removal are coordinated in bacteriophage T4 replication is different from that used in eukaryotes.The phage T4 member of the FEN1 nuclease family was called T4 RNase H because it hydrolyzed the RNA strand in an RNA:DNA hybrid, as expected for an enzyme removing the RNA primers. However, it also acts as a 5Ј-nuclease on DNA duplexes (3). Genetic studies indicate that either T4 RNase H or the 5Ј-to 3Ј-exonuclease of the Escherichia coli host DNA polymerase I is necessary for phage production (4). A T4 mutant with a large deletion (⌬118 -305) in the rnh gene gives a burst size of 50% of wild type T...

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Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication

Cited by 23 publications

References 26 publications

Bacteriophage T4 RNase H Removes Both RNA Primers and Adjacent DNA from the 5′ End of Lagging Strand Fragments

Bacteriophage T4 RNase H Removes Both RNA Primers and Adjacent DNA from the 5′ End of Lagging Strand Fragments

Bacteriophage T4 Genome

Maturation of Bacteriophage T4 Lagging Strand Fragments Depends on Interaction of T4 RNase H with T4 32 Protein Rather than the T4 Gene 45 Clamp

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