A new deoxyribonuclease activity from bacteria infected with T5 bacteriophage

Fielding, Phoebe E.; Lunt, Mary R.

doi:10.1016/0014-5793(69)80335-2

Cited by 7 publications

(5 citation statements)

References 8 publications

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“…A1 mutants do not degrade host DNA, suggesting perhaps a nuclease activity. However, apart from one preliminary report (25), no nuclease activity has been reported for the A1 protein or any other pre-early protein. One possibility, discussed in detail below, is that A1 codes for a site-specific restriction endonuclease.…”

Section: 22mentioning

confidence: 79%

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Phage T5 two-step injection

Davison

2019

Preprint

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Escherichia coli bacteriophage T5 differs from most phages in that it injects its genome in two steps: First Step Transfer, FST, corresponding to leftmost 7.9% of the genome and Second Step Transfer, SST, corresponding to the remainder of the genome. Expression of genes A1 and A2 is required for SST. DNA injection stops at a site known as the injection stop signal (iss) which is a cis acting site located in the untranslated region of the Left Terminal Repeat (LTR). The iss region is extremely complicated with many repeats, inverted repeats and palindromes. This report compares the iss regions of 21 T5 related phages and shows that all have a common conserved structure including a series of 8 DnaA boxes arranged in a highly specific manner; reminiscent of the origin of replication (oriC) of the host. DnaA protein, which binds DnaA boxes is a mostly membrane bound, leading to the suggestion that injection stops at iss due to binding to DnaA protein at the membrane. A new model of the mechanism of T5 injection stop and SST injection re-start is suggested.

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Section: 22mentioning

confidence: 79%

“…A2 protein is also needed, together with A1 protein, for SST transfer. A2 polypeptide has been purified and shown to be a DNA binding dimer (25). The A1 and A2 proteins are known to bind together and to be membrane associated.…”

Section: The Role Of A2 Proteinmentioning

confidence: 99%

Phage T5 two-step injection

Davison

2019

Preprint

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“…These four endonucleases are clearly distinguished from the only other reported T5-induced endonuclease (23), which makes doublestrand scissions on X DNA, T5 DNA, and T2 DNA. donuclease has a pronounced preference for sites in the "minus" strand of OX174 replicative form DNA, but shows no activity on viral q5X174 DNA (27).…”

Section: Resultsmentioning

confidence: 98%

Bacteriophage T5-induced endonucleases that introduce site-specific single-chain interruptions in duplex DNA.

Rogers¹,

Rhoades²

1976

Proc. Natl. Acad. Sci. U.S.A.

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Four site-specific endodeoxyribonucleases have been partially purified from extracts of bacteriophage T5-infected Escherichia coli by gel filtration and affinity chromatography on single-and double-stranded DNA. The enzymes were detected and characterized by agarose gel electrophoresis of alkali-denatured digestion products. None Hayward and Smith (8,9), using gel electrophoresis of denatured T5 DNA, were able to detect over 40 discrete classes of single-strand fragments that occur in various proportions. These classes are altered in a deletion mutant of T5 that lacks one of the interruptions (7, 9), which suggests that DNA site recognition is important in determining the location of the interruptions.On the assumption that T5-induced proteins were responsible for the interruptions, we prepared extracts from T5-infected cells and examined them for site-specific endonuclease activity. This paper reports the discovery and partial characterization of four such activities found in T5-infected cells. Horizontal 0.7% agarose slab gels were formed from agarose (Sigma) prepared by refluxing in Tris-phosphate buffer (8). Each gel was 15 X 9 X 0.7 cm thick and contained slots formed with a Plexiglas comb with 1.5 X 3 mm teeth. Wicks were cut from Whatman 3 MM paper. Following the loading of up to 27 samples, electrophoresis was begun at 120 V, constant voltage. After 20 min, the buffer was removed from the slots which were then filled with 0.7% agarose in Tris-phosphate. Electrophoresis was resumed at 120 V and continued for 6-7 hr at room temperature. At the end of a run, the gel was stained in 0.5 Mg/ml of ethidium bromide (Sigma), and a photograph was taken using the method of Sharp et al. (17) The pellet was frozen and stored at -200. MATERIALS AND METHODSExtracts were prepared by resuspending 3 g of infected cells in 12 ml of buffer A (20 mM Tris-HCl at pH 7.6, 1.0 M NaCl, 10 mM 2-mercaptoethanol). This and all subsequent steps were performed at 0-4°. The resuspended cells were disrupted by sonication and centrifuged at 27,000 X g for 15 min. The supernatant was then centrifuged in a Spinco 40 rotor at 35,000 rpm for 3 hr. The resultant supernatant (10 ml, 15 mg of protein per ml) was then chromatographed on a Bio-Gel A-0.5m (200-400 mesh) column equilibrated in buffer A. The flow rate was 18 ml/hr, and 4 ml fractions were collected. This step re-1576

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“…Analysis of the phosphomonoester type and the sizes of the products generated by A. mellea nuclease DNA partly degraded by A. mellea nuclease was rendered totally acid-soluble by subsequent treatment with snake-venom phosphodiesterase, which is a 3h exonuclease acting only on free 3h-OH groups [24]. Similarly, λ exonuclease, which degrades DNA strands from the 5h end only if a 5h-P group is present [25], also completely hydrolysed the products of A. mellea nuclease digestion (results not shown). This supports the conclusion that the enzyme cuts DNA molecules leaving 5h-P and 3h-OH termini on its products.…”

Section: Figure 5 Analysis Of Directionality Of Exonuclease Activity mentioning

confidence: 99%

Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea

Healy

Doonan²,

McCarthy³

1999

Biochemical Journal

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We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography. The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis. The nuclease was active on both double-stranded and single-stranded DNA but not on RNA. It was optimally active at pH8.5 and also exhibited a significant degree of thermostability. Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3'-5' (but not a 5'-3') exonuclease activity. It generated 5'-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-based approach. Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA. A BLAST search of the SwissProt database revealed that A. mellea nuclease shared significant amino acid similarity with two nucleases from Bacillus subtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes.

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A new deoxyribonuclease activity from bacteria infected with T5 bacteriophage

Cited by 7 publications

References 8 publications

Phage T5 two-step injection

Phage T5 two-step injection

Bacteriophage T5-induced endonucleases that introduce site-specific single-chain interruptions in duplex DNA.

Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea

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