Cyclin a Activates the DNA Polymerase δ -Dependent Elongation Machinery in vitro: A Parvovirus DNA Replication Model

Replication of the single-stranded linear DNA genome of parvovirus minute virus of mice (MVM) starts with complementary strand synthesis from the 3′-terminal snap-back telomere, which serves as a primer for the formation of double-stranded replicative form (RF) DNA. This DNA elongation reaction, des...

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-05, Vol.97 (10), p.5522-5527
Main Authors: Bashir, Tarig, Horlein, Rita, Rommelaere, Jean, Willwand, Kurt
Format: Article
Language:English
Subjects:
Animals
Biological Sciences
Cell cycle
Cell Cycle - physiology
Cell extracts
Cell Line
Conversion disorder
Cyclin A - genetics
Cyclin A - metabolism
Cyclin-Dependent Kinase Inhibitor p21
Cyclins
Cyclins - metabolism
Cytosol - metabolism
Deoxyribonucleic acid
DNA
DNA Polymerase III - metabolism
DNA Replication
Enzyme Inhibitors - metabolism
Fibroblasts
G1 Phase
Genomes
Genomics
Glutathione Transferase - genetics
Glutathione Transferase - metabolism
Histones
Interphase
Kinetics
Mice
Microbiology
Minute Virus of Mice - enzymology
Minute Virus of Mice - genetics
Parvovirus
Recombinant Fusion Proteins - metabolism
Rodents
S Phase
Spodoptera
Transfection
Virion - enzymology
Virion - genetics
Viruses
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Replication of the single-stranded linear DNA genome of parvovirus minute virus of mice (MVM) starts with complementary strand synthesis from the 3′-terminal snap-back telomere, which serves as a primer for the formation of double-stranded replicative form (RF) DNA. This DNA elongation reaction, designated conversion, is exclusively dependent on cellular factors. In cell extracts, we found that complementary strand synthesis was inhibited by the cyclin-dependent kinase inhibitor p21WAF1/CIP1and rescued by the addition of proliferating cell nuclear antigen, arguing for the involvement of DNA polymerase (Pol) δ in the conversion reaction. In vivo time course analyses using synchronized MVM-infected A9 cells allowed initial detection of MVM RF DNA at the G1/S phase transition, coinciding with the onset of cyclin A expression and cyclin A-associated kinase activity. Under in vitro conditions, formation of RF DNA was efficiently supported by A9 S cell extracts, but only marginally by G1cell extracts. Addition of recombinant cyclin A stimulated DNA conversion in G1cell extracts, and correlated with a concomitant increase in cyclin A-associated kinase activity. Conversely, a specific antibody neutralizing cyclin A-dependent kinase activity, abolished the capacity of S cell extracts for DNA conversion. We found no evidence for the involvement of cyclin E in the regulation of the conversion reaction. We conclude that cyclin A is necessary for activation of complementary strand synthesis, which we propose as a model reaction to study the cell cycle regulation of the Pol δ -dependent elongation machinery.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.090485297