Primase from escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins

Template sequence requirements based on the bacteriophage G4 complementary strand origin and okazaki fragment initiation sites

John R. Swart, Mark A Griep

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

We find that neither the hairpins nor bound single-stranded DNA-binding protein (SSB) are required for in vitro primase activity at the G4 complementary strand origin. Primase has the ability to prime synthetic DNA templates in the absence of any auxiliary proteins, including SSB, and in the absence of any DNA secondary structure. Primase does, however, initiate primer synthesis starting at a specific nucleotide sequence present in both the G4 origin system and at sites of Okazaki fragment initiation. We tested a series of single-stranded DNA templates that consisted of various portions of the G4 complementary strand origin for the ability to support primer synthesis. Primer synthesis activity was determined using an assay in which primer length and quantity are monitored by the incorporation of [γ-32P]ATP once per primer and the products analyzed on a denaturing polyacrylamide gel. We find that primase requires only a short DNA template, 5′-(AC)7CTG CAA AGC-3′, to synthesize a 17-nucleotide primer starting at the thymidine residue. We also report primase's ability to incorporate dideoxyribonucleotide triphosphates into primers, which has allowed us to determine directly both length and sequence of primers synthesized.

Original languageEnglish (US)
Pages (from-to)12970-12976
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number17
StatePublished - Jun 15 1993

Fingerprint

Microvirus
DNA Primase
Bacteriophages
DNA-Binding Proteins
Escherichia coli
Proteins
DNA
Nucleotides
Single-Stranded DNA
Thymidine
Assays
Thermodynamic properties
Adenosine Triphosphate
Okazaki fragments

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{adc179f93201460ea9a0cb2502abb754,
title = "Primase from escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins: Template sequence requirements based on the bacteriophage G4 complementary strand origin and okazaki fragment initiation sites",
abstract = "We find that neither the hairpins nor bound single-stranded DNA-binding protein (SSB) are required for in vitro primase activity at the G4 complementary strand origin. Primase has the ability to prime synthetic DNA templates in the absence of any auxiliary proteins, including SSB, and in the absence of any DNA secondary structure. Primase does, however, initiate primer synthesis starting at a specific nucleotide sequence present in both the G4 origin system and at sites of Okazaki fragment initiation. We tested a series of single-stranded DNA templates that consisted of various portions of the G4 complementary strand origin for the ability to support primer synthesis. Primer synthesis activity was determined using an assay in which primer length and quantity are monitored by the incorporation of [γ-32P]ATP once per primer and the products analyzed on a denaturing polyacrylamide gel. We find that primase requires only a short DNA template, 5′-(AC)7CTG CAA AGC-3′, to synthesize a 17-nucleotide primer starting at the thymidine residue. We also report primase's ability to incorporate dideoxyribonucleotide triphosphates into primers, which has allowed us to determine directly both length and sequence of primers synthesized.",
author = "Swart, {John R.} and Griep, {Mark A}",
year = "1993",
month = "6",
day = "15",
language = "English (US)",
volume = "268",
pages = "12970--12976",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Primase from escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins

T2 - Template sequence requirements based on the bacteriophage G4 complementary strand origin and okazaki fragment initiation sites

AU - Swart, John R.

AU - Griep, Mark A

PY - 1993/6/15

Y1 - 1993/6/15

N2 - We find that neither the hairpins nor bound single-stranded DNA-binding protein (SSB) are required for in vitro primase activity at the G4 complementary strand origin. Primase has the ability to prime synthetic DNA templates in the absence of any auxiliary proteins, including SSB, and in the absence of any DNA secondary structure. Primase does, however, initiate primer synthesis starting at a specific nucleotide sequence present in both the G4 origin system and at sites of Okazaki fragment initiation. We tested a series of single-stranded DNA templates that consisted of various portions of the G4 complementary strand origin for the ability to support primer synthesis. Primer synthesis activity was determined using an assay in which primer length and quantity are monitored by the incorporation of [γ-32P]ATP once per primer and the products analyzed on a denaturing polyacrylamide gel. We find that primase requires only a short DNA template, 5′-(AC)7CTG CAA AGC-3′, to synthesize a 17-nucleotide primer starting at the thymidine residue. We also report primase's ability to incorporate dideoxyribonucleotide triphosphates into primers, which has allowed us to determine directly both length and sequence of primers synthesized.

AB - We find that neither the hairpins nor bound single-stranded DNA-binding protein (SSB) are required for in vitro primase activity at the G4 complementary strand origin. Primase has the ability to prime synthetic DNA templates in the absence of any auxiliary proteins, including SSB, and in the absence of any DNA secondary structure. Primase does, however, initiate primer synthesis starting at a specific nucleotide sequence present in both the G4 origin system and at sites of Okazaki fragment initiation. We tested a series of single-stranded DNA templates that consisted of various portions of the G4 complementary strand origin for the ability to support primer synthesis. Primer synthesis activity was determined using an assay in which primer length and quantity are monitored by the incorporation of [γ-32P]ATP once per primer and the products analyzed on a denaturing polyacrylamide gel. We find that primase requires only a short DNA template, 5′-(AC)7CTG CAA AGC-3′, to synthesize a 17-nucleotide primer starting at the thymidine residue. We also report primase's ability to incorporate dideoxyribonucleotide triphosphates into primers, which has allowed us to determine directly both length and sequence of primers synthesized.

UR - http://www.scopus.com/inward/record.url?scp=0027160455&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027160455&partnerID=8YFLogxK

M3 - Article

VL - 268

SP - 12970

EP - 12976

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -