Resolution and purification of free primase activity from the DNA primase-poivmerase α complex of hela cells

Jamboor K. Vishwanatha, Earl F. Baril

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

DNA primase activlty has been resolved from a purified DNA primase-polymerase α complex of HeLa cells by hydrophobia affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase α. The free DMA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyaorylanide gel electrophoretio analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velcity sedimentation has an S20, W of 5. DNA primase synthesizes RNA ollgomers with single- stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase α in a manner that has already been described for several purified eukaryotic DNA primase-polymerase α complexes. The purified free DNA prinase activity is resistant to neutralizing anti-human DNA polymerase α antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase α and also DNA polymerase α complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase α. Taken together these results indicate that the DNA primase and polymerase α activities of the DNA primase-polymerase α complex reside on separate polypeptides that associate tightly through hydrophobio Interactions.

Original languageEnglish
Pages (from-to)8467-8487
Number of pages21
JournalNucleic Acids Research
Volume14
Issue number21
DOIs
StatePublished - 11 Nov 1986

Fingerprint

DNA Primase
HeLa Cells
DNA-Directed DNA Polymerase
Poly T
Aphidicolin
Rabies
DNA
Affinity Chromatography
Sodium Dodecyl Sulfate
Chromatography

Cite this

@article{01a10664cdb04db2a4acadde78cadfda,
title = "Resolution and purification of free primase activity from the DNA primase-poivmerase α complex of hela cells",
abstract = "DNA primase activlty has been resolved from a purified DNA primase-polymerase α complex of HeLa cells by hydrophobia affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55{\%}) of DNA primase that is free from polymerase α. The free DMA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyaorylanide gel electrophoretio analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velcity sedimentation has an S20, W of 5. DNA primase synthesizes RNA ollgomers with single- stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase α in a manner that has already been described for several purified eukaryotic DNA primase-polymerase α complexes. The purified free DNA prinase activity is resistant to neutralizing anti-human DNA polymerase α antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase α and also DNA polymerase α complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase α. Taken together these results indicate that the DNA primase and polymerase α activities of the DNA primase-polymerase α complex reside on separate polypeptides that associate tightly through hydrophobio Interactions.",
author = "Vishwanatha, {Jamboor K.} and Baril, {Earl F.}",
year = "1986",
month = "11",
day = "11",
doi = "10.1093/nar/14.21.8467",
language = "English",
volume = "14",
pages = "8467--8487",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "21",

}

Resolution and purification of free primase activity from the DNA primase-poivmerase α complex of hela cells. / Vishwanatha, Jamboor K.; Baril, Earl F.

In: Nucleic Acids Research, Vol. 14, No. 21, 11.11.1986, p. 8467-8487.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Resolution and purification of free primase activity from the DNA primase-poivmerase α complex of hela cells

AU - Vishwanatha, Jamboor K.

AU - Baril, Earl F.

PY - 1986/11/11

Y1 - 1986/11/11

N2 - DNA primase activlty has been resolved from a purified DNA primase-polymerase α complex of HeLa cells by hydrophobia affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase α. The free DMA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyaorylanide gel electrophoretio analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velcity sedimentation has an S20, W of 5. DNA primase synthesizes RNA ollgomers with single- stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase α in a manner that has already been described for several purified eukaryotic DNA primase-polymerase α complexes. The purified free DNA prinase activity is resistant to neutralizing anti-human DNA polymerase α antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase α and also DNA polymerase α complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase α. Taken together these results indicate that the DNA primase and polymerase α activities of the DNA primase-polymerase α complex reside on separate polypeptides that associate tightly through hydrophobio Interactions.

AB - DNA primase activlty has been resolved from a purified DNA primase-polymerase α complex of HeLa cells by hydrophobia affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase α. The free DMA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyaorylanide gel electrophoretio analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velcity sedimentation has an S20, W of 5. DNA primase synthesizes RNA ollgomers with single- stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase α in a manner that has already been described for several purified eukaryotic DNA primase-polymerase α complexes. The purified free DNA prinase activity is resistant to neutralizing anti-human DNA polymerase α antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase α and also DNA polymerase α complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase α. Taken together these results indicate that the DNA primase and polymerase α activities of the DNA primase-polymerase α complex reside on separate polypeptides that associate tightly through hydrophobio Interactions.

UR - http://www.scopus.com/inward/record.url?scp=0023047186&partnerID=8YFLogxK

U2 - 10.1093/nar/14.21.8467

DO - 10.1093/nar/14.21.8467

M3 - Article

C2 - 3786132

AN - SCOPUS:0023047186

VL - 14

SP - 8467

EP - 8487

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 21

ER -