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Nucleic Acids Research 2005 33(8):2410-2420; doi:10.1093/nar/gki539
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Published online 28 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

The requirement of yeast replication origins for pre-replication complex proteins is modulated by transcription

Conrad A. Nieduszynski, J. Julian Blow1 and Anne D. Donaldson*

Institute of Medical Sciences, University of Aberdeen Foresterhill, Aberdeen AB25 2ZD, Scotland, UK 1Cancer Research UK Chromosome Replication Research Group, Wellcome Trust Biocentre, University of Dundee Dow Street, Dundee DD1 5EH, Scotland, UK

*To whom correspondence should be addressed. Tel: +44 0 1224 550975; Fax: +44 0 1224 555844; Email: a.d.donaldson{at}abdn.ac.uk

Received January 12, 2005. Revised April 11, 2005. Accepted April 11, 2005.

The mini-chromosome maintenance proteins Mcm2–7 are essential for DNA replication. They are loaded onto replication origins during G1 phase of the cell cycle to form a pre-replication complex (pre-RC) that licenses each origin for subsequent initiation. We have investigated the DNA elements that determine the dependence of yeast replication origins on Mcm2–7 activity, i.e. the sensitivity of an origin to mcm mutations. Using chimaeric constructs from mcm sensitive and mcm insensitive origins, we have identified two main elements affecting the requirement for Mcm2–7 function. First, transcription into an origin increases its dependence on Mcm2–7 function, revealing a conflict between pre-RC assembly and transcription. Second, sequence elements within the minimal origin influence its mcm sensitivity. Replication origins show similar differences in sensitivity to mutations in other pre-RC proteins (such as Origin Recognition Complex and Cdc6), but not to mutations in initiation and elongation factors, demonstrating that the mcm sensitivity of an origin is determined by its ability to establish a pre-RC. We propose that there is a hierarchy of replication origins with respect to the range of pre-RC protein concentrations under which they will function. This hierarchy is both ‘hard-wired’ by the minimal origin sequences and ‘soft-wired’ by local transcriptional context.


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