Nucleotide sequence analysis of DNA. XXIV. Synchronous digestion of SV40 DNA by exonuclease III

Wü, Ray; Ruben, George C.; Siegel, Benjamin M.; Jay, Ernest; Spielman, Paul; Tu, Chen‐Pei D.

doi:10.1021/bi00649a003

Cited by 65 publications

(20 citation statements)

References 18 publications

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“…Furthermore, Yajko and Weiss (1975) have shown that point mutations in the xth structural gene led to a concomitant loss of both the endonucleolytic and exonucleolytic activities, suggesting that both the endonucleolytic and exonucleolytic activities may be sharing the same active site. Therefore, it has been assumed A .…”

Section: Mechanism Of Action Of Exonuclease III a Modifiedmentioning

confidence: 99%

Mechanism of action of Escherichia coli exonuclease III

Kow¹

1989

Biochemistry

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Exonuclease III is the major apurinic/apyrimidinic (AP) endonuclease of Escherichia coli, accounting for more than 80% of the total cellular AP endonuclease activity. We have shown earlier that the endonucleolytic activity of exonuclease III is able to hydrolyze the phosphodiester bond 5' to the urea N-glycoside in a duplex DNA [Kow, Y. W., & Wallace, S. S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8354-8358]. Therefore, we were interested in studying the mechanism of action of the endonucleolytic activity of exonuclease III by preparing DNA containing different base lesions as well as chemically modified AP sites. When AP sites were converted to O-alkylhydroxylamine residues, exonuclease III was able to hydrolyze the phosphodiester bond 5' to O-alkylhydroxylamine residues. The apparent Km for different O-alkylhydroxylamine residues was not affected by the particular O-alkylhydroxylamine residue substituted; however, the apparent Vmax decreased as the size of the residue increased. On the basis of a study of the substrate specificity of exonuclease III, a modification of the Weiss model for the mechanism of action of exonuclease III is presented. Furthermore, a temperature study of exonucleolytic activity of exonuclease III in the presence of Mg2+ showed discontinuity in the Arrhenius plot. However, no discontinuity was observed when the reaction was performed in the presence of Ca2+. Similarly, no discontinuity was observed for the endonucleolytic activity of exonuclease III, in the presence of either Ca2+ or Mg2+. These data suggest that, in the presence of Mg2+, exonuclease III, in the presence of either Ca2+ or Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Mechanism Of Action Of Exonuclease III a Modifiedmentioning

confidence: 99%

Mechanism of action of Escherichia coli exonuclease III

Kow¹

1989

Biochemistry

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“…Two different schemes are currently employed for progressively varying the length of double-stranded DNA fragments. One is the use of exonuclease III followed by single-strand-specific S1 nuclease (1,2,4,5,7,8). Another scheme uses Bal 31 nuclease (3, 6), which simultaneously digests both 5' and 3' ends of a doublestranded DNA fragment (9).…”

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confidence: 99%

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

Putney¹,

Benkovic²,

Schimmel³

1981

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

180

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2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP [caS]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to. be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand ofa double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease mI is limited and is followed by SI nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this way thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and SI nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.It is often useful to decrease the length ofa given DNA fragment in a progressive, controlled manner (1-7). Two different schemes are currently employed for progressively varying the length of double-stranded DNA fragments. One is the use of exonuclease III followed by single-strand-specific S1 nuclease (1,2,4,5,7,8). Another scheme uses Bal 31 nuclease (3, 6), which simultaneously digests both 5' and 3' ends of a doublestranded DNA fragment (9). The primary disadvantage of these approaches is that the nucleases act simultaneously at both ends of the fragment so that desirable sequences at one end are not preserved.The Sp diastereomer of the nucleoside a-thiotriphosphates (which have a sulfur substituted for an oxygen at the a-phosphate) act as substrates for Escherichia coli DNA polymerase I in DNA synthesis (10). These compounds, although incorporated at an efficiency similar to that of the natural substrates, effectively block the 3' exonuclease activity of DNA polymerase I at the phosphorothioate nucleotide linkage (unpublished results). In work reported here we found that placement of an athionucleotide at only one of the 3' ends of a DNA fragment effectively blocks that end from digestion with exonuclease III. Digestion proceeds progressively from the opposite end and, after S1 nuclease treatment, the fragment is shortened with the integrity of the nucleotide sequence at the "capped" end preserved. Alternatively, if exonuclease III digestion is allowed to proceed to completion, a full-length single strand is generated from the original double strand.This work also demonstrates that, when 2'-deoxyadenosine 5'-O-(l-thiotriphosphate) (dATP [aS]) is inserted into 3' recessed ends ofrestriction fragments, the phosphorothioate linkage allows subsequent ligation ofthe DNA, restriction ofligated junctions, and in vivo replication of plasmids containing phosphorothioate nucleotide linkages. Purific...

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“…It had been determined that Exo III digested in processive and synchronous (when added in excess) manners (17) and was demonstrated to remove up to 14.4 kb DNA sequence during 32 min incubation at 37°C (18). However, phosphorothioate linkages were resistant to Exo III (16) which should have ensured the ''safety'' of Oligo1-ligated template DNA.…”

Section: Resultsmentioning

confidence: 99%