Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture

Conrad A Nieduszynski,Calvin Tiengwe,Richard Mcculloch,Stephen D Bell,Helen Farr,Richard Burchmore,Catarina Gadelha,J David Barry,Lucio Marcello

doi:10.1371/journal.pone.0032674

Abstract

DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

Highlights

Genome replication is central to the propagation of all life
The T. brucei MCM helicase is a conserved heterohexamer In eukaryotes Cdc6 and Cdt1 function to recruit the MCM
A mechanistic consequence of such a putative absence could be that MCM in T. brucei is recruited directly by TbORC1/ CDC6

Summary

Introduction

Genome replication is central to the propagation of all life. In DNA genomes, replication initiates by the designation of genome sequences as origins, where synthesis of a copy of the genetic material begins. Origin designation is a complex, tightly regulated process whose core mechanisms and machinery are conserved between eukaryotes and archaea [1,2] This reaction involves the formation of a pre-replication complex (pre-RC), which in eukaryotes comprises the Origin Recognition Complex (ORC), Cdc, Cdt and the replicative MCM helicase; for reviews, see [3,4,5,6]. ORC is frequently described as being composed of six subunits, named Orc, in all eukaryotes that have been examined to date [7]. It is the earliest acting pre-RC component during DNA replication origin designation, being responsible for binding to origins. ATP binding and hydrolysis by the ORC subunits causes conformational changes associated with ORC assembly and DNA binding, as well as modulating interaction with the other pre-RC components [12,15,16,17,18]

Methods

Results

Conclusion