RNAP-II Molecules Participate in the Anchoring of the ORC to rDNA Replication Origins

Maria D Mayan,M Carolina Elias

doi:10.1371/journal.pone.0053405

Abstract

The replication of genomic DNA is limited to a single round per cell cycle. The first component, which recognises and remains bound to origins from recognition until activation and replication elongation, is the origin recognition complex. How origin recognition complex (ORC) proteins remain associated with chromatin throughout the cell cycle is not yet completely understood. Several genome-wide studies have undoubtedly demonstrated that RNA polymerase II (RNAP-II) binding sites overlap with replication origins and with the binding sites of the replication components. RNAP-II is no longer merely associated with transcription elongation. Several reports have demonstrated that RNAP-II molecules affect chromatin structure, transcription, mRNA processing, recombination and DNA repair, among others. Most of these activities have been reported to directly depend on the interaction of proteins with the C-terminal domain (CTD) of RNAP-II. Two-dimensional gels results and ChIP analysis presented herein suggest that stalled RNAP-II molecules bound to the rDNA chromatin participate in the anchoring of ORC proteins to origins during the G1 and S-phases. The results show that in the absence of RNAP-II, Orc1p, Orc2p and Cdc6p do not bind to origins. Moreover, co-immunoprecipitation experiments suggest that Ser2P-CTD and hypophosphorylated RNAP-II interact with Orc1p. In the context of rDNA, cryptic transcription by RNAP-II did not negatively interfere with DNA replication. However, the results indicate that RNAP-II is not necessary to maintain the binding of ORCs to the origins during metaphase. These findings highlight for the first time the potential importance of stalled RNAP-II in the regulation of DNA replication.

Highlights

Replication originates at multiple and specific locations in the genome that define starting sites, which are called replication origins (ORIs) [1]
Orc1p binding tended to increase in the absence of RPB1. The aim of this project was to study whether RNA polymerase II (RNAP-II) influences the activity of the ORIs in the rDNA locus of S. cerevisiae
We have previously reported that stalled RNAP-II complexes at the rDNA locus contain the typical form of stalled RNAP-II phosphorylated in the residue Ser5 of its C-terminal domain (CTD) [38]

Summary

Introduction

Replication originates at multiple and specific locations in the genome that define starting sites, which are called replication origins (ORIs) [1]. The initiation of chromosomal DNA replication is a complex process that assures that every replication origin fires once and only once during the S-phase. (a) The origins are recognised by the pre-replication complex (pre-RC), which is a process called licensing and is responsible for recruiting additional replication factors to this site. The pre-RC typically forms at the end of mitosis by the binding of origin recognition complex (ORC) [11] to chromosomal DNA, which enables the interaction of the replication proteins, which are Cdc, Cdt and six MCM subunits (Mcm). Pre-RC formation renders the chromatin competent for replication and is restricted to the G1 phase of the cell cycle, forming the so-called ‘‘window of opportunity’’. (b) The second step in the triggering of origin activation is the recruitment of additional initiator proteins to the pre-RC to form the pre-initiation complex (pre-IC). Pre-RC formation renders the chromatin competent for replication and is restricted to the G1 phase of the cell cycle, forming the so-called ‘‘window of opportunity’’. (b) The second step in the triggering of origin activation is the recruitment of additional initiator proteins to the pre-RC to form the pre-initiation complex (pre-IC). (c) DNA synthesis initiates after an activation step

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