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Biochemical properties of the stimulatory activity of DNA polymerase alpha by the hyper-phosphorylated retinoblastoma protein.

Authors
  • Takemura, Masaharu
Type
Published Article
Journal
Biochimica et Biophysica Acta
Publisher
Elsevier
Publication Date
Jun 06, 2002
Volume
1571
Issue
2
Pages
151–156
Identifiers
PMID: 12049795
Source
Medline
License
Unknown

Abstract

Previously, my colleagues and I have reported that the immunopurified hyper-phosphorylated retinoblastoma protein (ppRb) stimulates the activity of DNA polymerase alpha. I describe here the biochemical characteristics of this stimulatory activity. DNA polymerase alpha-stimulatory activity of ppRb was most remarkable when using activated DNA as a template-primer, rather than using poly(dT)-(rA)(10), poly(dA)-(dT)(12-18), and so on. Kinetic analysis showed that there was no significant difference in K(m) value for deoxyribonucleotides of DNA polymerase alpha in the presence of ppRb. Adding ppRb resulted in the overcoming pause site on the template, but did not affect the rate of misincorporation of incorrect deoxyribonucleotides. By adding ppRb, the optimal concentration of template-primer was shifted to a higher region, but not using M13 singly primed DNA. The ppRb seemed to assist the process that DNA polymerase alpha changed its conformation resulting in appropriate enzyme activity. These results suggest that ppRb affects both template-primer and DNA polymerase alpha and makes appropriate circumstances for the enzyme reaction.

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