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.
N1 - Funding Information:
ACKNOWLEDGMENTS The contributions of Dr. Mary Hesolowski-Owen and Paul Bonln in the early phase of this study is gratefully acknowledged. We thank Dr. Samuel Wadsworth for helpful disousalons in the gel eleotrophoretio analysis of the DNA and RNA produota. We are grateful to Ms. Sandra Johnson and Lois Hager for their expert assistance in preparing the manuscript. A preliminary version of these results was presented at the 13th International Bioohemical Congress in Amsterdam, The Netherlands, August 1985. This work was supported by Public Health Service Grants P30-12708 and CA15187 from the National Institutes of Health. JKV is a Fellow of The Leukemia Society of America.
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
SN - 0305-1048
VL - 14
SP - 8467
EP - 8487
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 21
ER -