Uncoupling of Unwinding From DNA Synthesis Implies Regulation of MCM Helicase by Tof1/Mrc1/Csm3 Checkpoint Complex

J Mol Biol. 2005 Apr 1;347(3):509-21. doi: 10.1016/j.jmb.2005.01.041.

Abstract

The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle / physiology
  • Cell Cycle Proteins / metabolism*
  • DNA Helicases / metabolism*
  • DNA Polymerase I / metabolism
  • DNA Replication*
  • DNA, Superhelical* / biosynthesis
  • DNA, Superhelical* / chemistry
  • DNA, Superhelical* / metabolism
  • DNA-Binding Proteins
  • Hydroxyurea / metabolism
  • Macromolecular Substances
  • Nucleic Acid Conformation
  • Plasmids / genetics
  • Plasmids / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • DNA, Superhelical
  • DNA-Binding Proteins
  • MRC1 protein, S cerevisiae
  • Macromolecular Substances
  • Saccharomyces cerevisiae Proteins
  • TOF1 protein, S cerevisiae
  • DNA Polymerase I
  • DNA Helicases
  • Hydroxyurea