Plk1 Phosphorylation of Orc2 Promotes DNA Replication under Conditions of Stress

Abstract

Polo-like kinase 1 (Plk1) plays pivotal roles in mitosis; however, little is known about its function in S phase. In this study, we show that inhibition of Plk1 impairs DNA replication and results in slow S-phase progression in cultured cancer cells. We have identified origin recognition complex 2 (Orc2), a member of the DNA replication machinery, as a Plk1 substrate and have shown that Plk1 phosphorylates Orc2 at Ser188 in vitro and in vivo. Furthermore, Orc2-S188 phosphorylation is enhanced when DNA replication is under challenge induced by ultraviolet, hydroxyurea, gemcitabine, or aphidicolin treatment. Cells expressing the unphosphorylatable mutant (S188A) of Orc2 had defects in DNA synthesis under stress, suggesting that this phosphorylation event is critical to maintain DNA replication under stress. To dissect the mechanism pertinent to this observation, we showed that Orc2-S188 phosphorylation associates with DNA replication origin and that cells expressing Orc2-S188A mutant fail to maintain the functional pre-replicative complex (pre-RC) under DNA replication stress. Furthermore, the intra-S-phase checkpoint is activated in Orc2-S188A-expressing cells to cause delay of S-phase progress. Our study suggests a novel role of Plk1 in facilitating DNA replication under conditions of stress to maintain genomic integrity.

ACKNOWLEDGMENTS

We thank Kevin Struhl from Harvard Medical School for sharing primers in ChIP assay and Benoit Miotto for discussions with ChIP techniques. We thank Scott Briggs, Paul South, and Hai-Ning Du from Purdue University for discussions of ChIP techniques. We thank Bruce Stillman for the T7-Orc2 construct. We appreciate Eleanor Erikson for critical reading of the manuscript.

X.L. is a recipient of the Howard Temin Award from the National Cancer Institute (K01CA114401 ). Support from the Purdue University Center for Cancer Research Small Grants Program is gratefully acknowledged. This project was also supported by National Science Foundation (MCB-1049693), the Elsa U. Pardee Foundation (grant 204937), and Uniting against Lung Cancer (grant 09107892).