Cyclin A activates the DNA polymerase δ-dependent elongation machinery in vitro : A parvovirus DNA replication model

Abstract

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 cyclindependent kinase inhibitor p21 WAF1/CIP1 and rescued by the addition of proliferating cell nuclear antigen, arguing for the involvement of DNA polymerase (Pol) ␦ in the conversion reaction. In vivo time course analyses using synchronized MVM-infected A9 cells allowed initial detection of MVM RF DNA at the G1͞S 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 G 1 cell extracts. Addition of recombinant cyclin A stimulated DNA conversion in G 1 cell 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 ␦-dependent elongation machinery. E ukaryotic DNA replication is restricted to the S phase of the cell cycle. Cell cycle progression is regulated through the action of cyclin-dependent kinases (cdks) that are sequentially activated on association with different cyclins (1). In vertebrate cells, the transition from G 1 to S phase is controlled by the activities of cyclin E-and cyclin A-dependent kinases (2). There is accumulating evidence for the involvement of cyclin A and cyclin E in the regulation of cellular DNA replication. Cyclin A is localized at nuclear replication foci in mammalian cells (3, 4), and both cyclin A and cdk2 have been found to be associated with replicating DNA in the simian virus 40 (SV40) in vitro replication system (5). Addition of recombinant cyclin A͞cdk2 and cyclin E͞cdk2 to G 1 phase nuclei has been shown to trigger the initiation of DNA replication in a human cell-free system (6). Similarly, the inability of human G 1 cell extracts to replicate SV40 origin-containing DNA in vitro could be overcome by the addition of cyclin A or active cdc2 kinase (7,8). Conversely, immunodepletion of cyclin A from S cell extracts partially inhibited SV40 origin-driven plasmid replication (9), whereas a significant decrease in the ability of Xenopus egg extracts to replicate sperm DNA was observed after depletion of cyclin E or cdk2 (10, 11). However, it has been difficult to identify replication factors that are targets for cdks in vivo a...

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