Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast

EMBO J. 2007 Mar 7;26(5):1327-39. doi: 10.1038/sj.emboj.7601585. Epub 2007 Feb 15.

Abstract

DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2'-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle / genetics
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Centromere / metabolism
  • Checkpoint Kinase 2
  • Chromosomes, Fungal
  • DNA Replication / drug effects
  • DNA Replication / genetics*
  • DNA, Intergenic / genetics
  • Dideoxynucleosides / metabolism
  • Electrophoresis, Gel, Two-Dimensional
  • Genome, Fungal*
  • Heterochromatin / metabolism
  • Hydroxyurea / pharmacology
  • Minichromosome Maintenance Complex Component 6
  • Oligonucleotide Array Sequence Analysis
  • Origin Recognition Complex / genetics
  • Origin Recognition Complex / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Replication Origin / genetics*
  • Schizosaccharomyces / drug effects
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces / metabolism
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism

Substances

  • Cell Cycle Proteins
  • DNA, Intergenic
  • Dideoxynucleosides
  • Heterochromatin
  • Origin Recognition Complex
  • Schizosaccharomyces pombe Proteins
  • 5-bromo-2',3'-dideoxyuridine
  • Checkpoint Kinase 2
  • Cds1 protein, S pombe
  • Protein-Serine-Threonine Kinases
  • Minichromosome Maintenance Complex Component 6
  • mcm6 protein, S pombe
  • Hydroxyurea