Gene 6 Exonuclease of Bacteriophage T7

Kerr, Carol; Sadowski, Paul D.

doi:10.1016/s0021-9258(19)45791-8

Cited by 105 publications

(12 citation statements)

References 31 publications

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“…Exo III degrades duplex DNA in the 3'-5' direction from 3'-OH or 3'-P04 termini, preferring recessed 3' ends over matched ends of duplex DNA or single phosphodiester bond interruptions (Rogers & Weiss, 1980). Exo T76 degrades duplex DNA in the 5-3' direction only from 5'-OH or 5'-P04 termini (including 5'-terminal RNA primers), preferring recessed 5' ends (Kerr & Sadowski, 1972;Shinozaki & Okazaki, 1978; M. Engler and C. C. Richardson, unpublished experiments). MNase is an endonuclease that degrades duplex or single-stranded DNA or RNA, preferring single strands at AT-rich regions (Anfinsen et al, 1971).…”

Section: Resultsmentioning

confidence: 95%

Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease

Cusick¹,

Herman²,

DePamphilis³

et al. 1981

Biochemistry

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Replicating simian virus 40 (SV40) chromosomes were found to be similar to other eukaryotic chromosomes in that the rate and extent of micrococcal nuclease (MNase) digestion were greater with replicating than with nonreplicating mature SV40 chromatin. MNase digestion of replicating SV40 chromosomes, pulse labeled in either intact cells or nuclear extracts, resulted in the rapid release of nascent DNA as essentially bare fragments of duplex DNA (3-7S) that had an average length of 120 base pairs and were degraded during the course of the reaction. In addition, nucleosomal monomers, equivalent in size to those from mature chromosomes, were released. On the other hand, MNase digestion of uniformly labeled mature SV40 chromosomes resulted in the release of only nucleosomal monomers and oligomers. The small nascent DNA fragments released from replicating chromosomes represented prenucleosomal DNA (PN-DNA) from the region of replication forks that encompasses the actual sites of DNA synthesis and includes Okazaki fragments. Predigestion of replicating SV40 chromosomes with both Escherichia coli exonuclease III (3'-5') and bacteriophage T7 gene 6 exonuclease (5'-3') resulted in complete degradation of PN-DNA. This result, together with the observation that isolated PN-DNA annealed equally well to both strands of SV40 restriction fragments, demonstrated that PN-DNA originates from both sides of replication forks. Over 90% of isolated Okazaki fragments annealed only to the retrograde DNA template. The characteristics of isolated PN-DNA were assessed by examining its sensitivity to MNase and single strand specific S1 endonuclease, sedimentation behavior before and after deproteinization, buoyant density in CsCl after formaldehyde treatment, and size on agarose gels. In addition, it was observed that MNase digestion of purified SV40 DNA also resulted in the release of a transient intermediate similar in size to PN-DNA, indicating that a DNA-protein complex is not required to account for the appearance of PN-DNA. These and other data provide a model of replicating chromosomes in which DNA synthesis occurs on a region of replication forks that is free of nucleosomes and is designated as prenucleosomal DNA.

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Section: Resultsmentioning

confidence: 95%

Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease

Cusick¹,

Herman²,

DePamphilis³

et al. 1981

Biochemistry

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“…9 T7 exonuclease cleaves double-stranded DNA in a 5′ → 3′ direction, removing 5′-mononucleotides from the 5′ termini or at gaps and nicks of double-stranded DNA. 10,11 A generic exonuclease reaction is shown in Figure 1.…”

Section: ■ Introductionmentioning

confidence: 99%

Analyzing Exonuclease-Induced Hyperchromicity by UV Spectroscopy: An Undergraduate Biochemistry Laboratory Experiment

et al. 2016

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An undergraduate biochemistry laboratory experiment is described that utilizes free online bioinformatics tools along with readily available exonucleases to study the effects of base stacking and hydrogen bonding on the UV absorbance of DNA samples. UV absorbance of double-stranded DNA at the λ max is decreased when the DNA bases are involved in hydrogen bonding and formation of secondary structure. When an exonuclease is added to the solution containing the DNA, the strand is digested and the interactions disappear, leading to an increase in the absorbance called hyperchromicity. This experiment utilizes exonuclease digestion of DNA to show students how base interactions and secondary structure can alter the spectroscopic properties of a sample and, by extension, how apparent concentration as calculated with Beer−Lambert's law is not necessarily representative of the true concentration of DNA in solution. Teaching applications of this laboratory experiment include enzyme kinetics and activity, secondary structure of single-stranded DNA (and its parallel to RNA structure), and an introduction to bioinformatics.

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“…In order to determine whether the effects of salt and DNA binding protein were occurring at the initial stage of exonucleolytic digestion, we examined the action of the exonuclease upon a DNA substrate labeled at its 5' termini with 32P by polynucleotide kinase. Since the exonuclease acts from 5' termini (Kerr & Sadowski, 1972b), the liberation of 32P radioactivity reflects the initiation of the reaction and the first phosphodiester bond cleavage. Since this reaction is extremely rapid, the experiments were done at 0 °C and with an 8-fold excess of DNA termini over enzyme molecules.…”

Section: Resultsmentioning

confidence: 99%

“…Inhibition of Exonuclease by NaCl Is Not Due to the 3' Single-Stranded Terminus. The S'-exonuclease activity of the gene 6 product generates a DNA molecule that has protruding 3' single-stranded tails (Kerr & Sadowski, 1972b). Since the presence of salt inhibits the gene 6 exonuclease, it might be thought that the salt might facilitate intrastrand base pairing in this tail that in turn might loop back on itself and sterically hinder access of exonuclease molecules to the recessed 5' terminus (see Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Roberts¹,

Sadowski²,

Wong³

1982

Biochemistry

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Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. The gene 6 exonuclease is a double-stranded DNA specific 5'-exonuclease that has been implicated in destruction of bacterial DNA, removal of RNA primers during DNA replication, genetic recombination, and DNA maturation. The enzyme is markedly inhibited by physiological concentrations of NaCl. This inhibition, which is due to a marked reduction in the Vmax of the enzyme, can be largely overcome by the phage-coded DNA binding protein. This stimulation is specific since the Escherichia coli DNA binding protein is without effect. The stimulation by the binding protein is apparently not due to its coating of the 3' single-stranded tails generated during the digestion. Kinetic studies show that the stimulation is due to a combined effect on both the Km and Vmax of the exonuclease. These studies are consistent with a loose binding of the binding protein to either the DNA or the exonuclease.

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Gene 6 Exonuclease of Bacteriophage T7

Cited by 105 publications

References 31 publications

Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease

Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease

Analyzing Exonuclease-Induced Hyperchromicity by UV Spectroscopy: An Undergraduate Biochemistry Laboratory Experiment

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

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