Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

Ramiro Mendoza-Maldonado,Roberta Paolinelli,Laura Galbiati,Mary Bryk,Mauro Giacca,Sara Giadrossi

doi:10.1371/journal.pone.0013720

Abstract

BackgroundThe retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition.Methodology/Principal FindingsHere we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border.Conclusions/SignificanceThe participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

Highlights

Fundamental work initially carried out in S. cerevisiae and extended to other organisms, has shown that origins of DNA replication are the sites at which the ordered assembly of the multiprotein pre-replicative complex takes place
retinoblastoma protein (Rb) and E2F1 proteins are recruited to human origins of DNA replication
Using a high resolution chromatin immunoprecipitation (ChIP) procedure, we have recently mapped the regions of binding of several components of the pre-replicative complex (pre-RC), including Orc1, Orc2, Mcm5 and CDC6, at these origins in close correspondence to the sites of nascent DNA synthesis [14]

Summary

Introduction

Fundamental work initially carried out in S. cerevisiae and extended to other organisms, has shown that origins of DNA replication are the sites at which the ordered assembly of the multiprotein pre-replicative complex (pre-RC) takes place. During the G1 phase of the cell cycle, the six-subunit origin recognition complex (ORC) is first recruited onto DNA; the CDC6 and Cdt proteins are required together for loading the putative replicative helicase (Mcm2-7) onto chromatin. Upon phosphorylation by the G1 phase CDKs, repression by Rb is relieved and transcription of the E2F-target genes ensues This is, an effective mechanism to couple cell cycle progression to the expression of genes that are required for DNA replication. The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition

Methods

Results

Conclusion