Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Proc Natl Acad Sci U S A. 2016 Jun 28;113(26):E3639-48. doi: 10.1073/pnas.1607552113. Epub 2016 Jun 13.


Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1-Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2-7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2-7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2-7 had to be phosphorylated for binding to phospho-Tof1-Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2-7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1-Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1-Ter complex.

Keywords: DDK; Fob1; Ter; Tof1; programmed fork arrest.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Replication*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Phosphorylation
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*


  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • TOF1 protein, S cerevisiae
  • CDC7 protein, S cerevisiae
  • Protein Serine-Threonine Kinases
  • DNA Helicases