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P21, an inhibitor of cyclin-dependent kinases, does not inhibit proliferating-cell nuclear antigen (PCNA)-dependent nucleotide-excision repair. P21 limitedly inhibits the short gap-filling DNA proliferation by PCNA-dependent DNA polymerases delta and epsilon more than the long primer-extension proliferation. P21 inhibition of PCNA's role in DNA duplication but not in DNA repair provides reason for the theory that genetic damage inactivates chromosomal replication but permits damage-induced repair.

Simian virus 40 DNA duplication indicates that p21 controls the proliferating cell nuclear antigen (PCNA)-based DNA duplications without a cyclin-dependent protein kinase. p21 prevents PCNA stimulation of DNA polymerase delta, the main replicative DNA polymerase which is due to a reaction between p21 and PCNA. p21 and PCNA are vital for cell-cycle development, DNA duplication and restructuring impaired DNA during p53-mediated restriction of cell proliferation.

Studies on a cell-free system developed to replicate DNA from SV40 established the identity of eukaryotic cell DNA replication machinery and the role of all the replication proteins. Biochemical fractionation of human cell extracts identified the cellular replication components that combine with SV40 large T antigen to replicate SV40 containing DNA. Now highly purified proteins replicate SV40 containing plasmid DNA including the two DNA polymerases alpha and delta.

DNA polymerase [Epsilon] (Pol[Epsilon]) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases [Alpha] and [Delta] (Pol[Alpha] and [Delta]), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol[Epsilon] in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol[Epsilon] is indeed required for chromosomal DNA replication. In Pol[Epsilon]-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol[Alpha] are loaded onto the chromatin template in Pol[Epsilon]-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol[Epsilon], along with Pol[Alpha] and Pol[Delta], is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

Background: DNA polymerase epsilon (Pol epsilon) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol alpha and Pol delta in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol epsilon in chromosomal DNA replication is not well understood., Results: This study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol epsilon. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol epsilon-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol epsilon holoenzyme to Pol epsilon-depleted extracts, but not by addition of polymerase-deficient forms of Pol epsilon, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol epsilon holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells., Conclusion: These results indicate that the DNA polymerase activity of Pol epsilon holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol epsilon holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts.