Completion of mammalian lagging strand DNA replication using purified proteins

John Turchi, Robert A. Bambara

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

We have modeled the reactions involved in the completion of lagging strand DNA replication using a synthetic DNA substrate and purified enzymes from calf thymus. We have demonstrated that each polymerase, α, δ, and ε, is capable of extending an upstream 3′ terminus to generate a nick in the DNA substrate that is subsequently ligated by DNA ligase I. Synthesis by each polymerase and subsequent ligation occurred efficiently after the addition of the 50-kDa 5′- to 3′-exonuclease. Analyses are presented which show that a substantial proportion of the ligated products is the result of polymerase ε, exonuclease, and DNA ligase I, all acting on the same DNA template. That is, polymerase fills in the gap, then both the polymerase and exonuclease act, one adding and the other removing nucleotides, followed by ligation. Results presented suggest that polymerase α, δ, or ε may functionally interact with DNA ligase I and the 5′- to 3′-exonuclease to perform the enzymatic reactions required for the completion of lagging strand DNA synthesis.

Original languageEnglish (US)
Pages (from-to)15136-15141
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number20
StatePublished - Jul 15 1993
Externally publishedYes

Fingerprint

Exonucleases
DNA Replication
spleen exonuclease
DNA Ligases
DNA
Ligation
Proteins
Single-Stranded DNA Breaks
Thymus
Thymus Gland
Substrates
Nucleotides
Enzymes
DNA Ligase ATP

ASJC Scopus subject areas

  • Biochemistry

Cite this

Completion of mammalian lagging strand DNA replication using purified proteins. / Turchi, John; Bambara, Robert A.

In: Journal of Biological Chemistry, Vol. 268, No. 20, 15.07.1993, p. 15136-15141.

Research output: Contribution to journalArticle

@article{aeda2c34283945719b059c4c2cb55d33,
title = "Completion of mammalian lagging strand DNA replication using purified proteins",
abstract = "We have modeled the reactions involved in the completion of lagging strand DNA replication using a synthetic DNA substrate and purified enzymes from calf thymus. We have demonstrated that each polymerase, α, δ, and ε, is capable of extending an upstream 3′ terminus to generate a nick in the DNA substrate that is subsequently ligated by DNA ligase I. Synthesis by each polymerase and subsequent ligation occurred efficiently after the addition of the 50-kDa 5′- to 3′-exonuclease. Analyses are presented which show that a substantial proportion of the ligated products is the result of polymerase ε, exonuclease, and DNA ligase I, all acting on the same DNA template. That is, polymerase fills in the gap, then both the polymerase and exonuclease act, one adding and the other removing nucleotides, followed by ligation. Results presented suggest that polymerase α, δ, or ε may functionally interact with DNA ligase I and the 5′- to 3′-exonuclease to perform the enzymatic reactions required for the completion of lagging strand DNA synthesis.",
author = "John Turchi and Bambara, {Robert A.}",
year = "1993",
month = "7",
day = "15",
language = "English (US)",
volume = "268",
pages = "15136--15141",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "20",

}

TY - JOUR

T1 - Completion of mammalian lagging strand DNA replication using purified proteins

AU - Turchi, John

AU - Bambara, Robert A.

PY - 1993/7/15

Y1 - 1993/7/15

N2 - We have modeled the reactions involved in the completion of lagging strand DNA replication using a synthetic DNA substrate and purified enzymes from calf thymus. We have demonstrated that each polymerase, α, δ, and ε, is capable of extending an upstream 3′ terminus to generate a nick in the DNA substrate that is subsequently ligated by DNA ligase I. Synthesis by each polymerase and subsequent ligation occurred efficiently after the addition of the 50-kDa 5′- to 3′-exonuclease. Analyses are presented which show that a substantial proportion of the ligated products is the result of polymerase ε, exonuclease, and DNA ligase I, all acting on the same DNA template. That is, polymerase fills in the gap, then both the polymerase and exonuclease act, one adding and the other removing nucleotides, followed by ligation. Results presented suggest that polymerase α, δ, or ε may functionally interact with DNA ligase I and the 5′- to 3′-exonuclease to perform the enzymatic reactions required for the completion of lagging strand DNA synthesis.

AB - We have modeled the reactions involved in the completion of lagging strand DNA replication using a synthetic DNA substrate and purified enzymes from calf thymus. We have demonstrated that each polymerase, α, δ, and ε, is capable of extending an upstream 3′ terminus to generate a nick in the DNA substrate that is subsequently ligated by DNA ligase I. Synthesis by each polymerase and subsequent ligation occurred efficiently after the addition of the 50-kDa 5′- to 3′-exonuclease. Analyses are presented which show that a substantial proportion of the ligated products is the result of polymerase ε, exonuclease, and DNA ligase I, all acting on the same DNA template. That is, polymerase fills in the gap, then both the polymerase and exonuclease act, one adding and the other removing nucleotides, followed by ligation. Results presented suggest that polymerase α, δ, or ε may functionally interact with DNA ligase I and the 5′- to 3′-exonuclease to perform the enzymatic reactions required for the completion of lagging strand DNA synthesis.

UR - http://www.scopus.com/inward/record.url?scp=0027201098&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027201098&partnerID=8YFLogxK

M3 - Article

VL - 268

SP - 15136

EP - 15141

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -