Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks

Nat Struct Mol Biol. 2020 May;27(5):461-471. doi: 10.1038/s41594-020-0407-7. Epub 2020 Apr 27.

Abstract

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase-polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Checkpoint Kinase 2 / genetics
  • Checkpoint Kinase 2 / metabolism*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Polymerase II / genetics
  • DNA Polymerase II / metabolism
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism
  • DNA Primers
  • DNA Replication*
  • DNA, Fungal / biosynthesis*
  • DNA, Fungal / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Mutation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Nucleosomes / genetics
  • Plasmids
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • CDC45 protein, S cerevisiae
  • Cell Cycle Proteins
  • DNA Primers
  • DNA, Fungal
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Nucleosomes
  • POL30 protein, S cerevisiae
  • Proliferating Cell Nuclear Antigen
  • Saccharomyces cerevisiae Proteins
  • Checkpoint Kinase 2
  • RAD53 protein, S cerevisiae
  • DNA Polymerase II
  • DNA Polymerase III
  • DNA Helicases