Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase

Fabrizio Villa; Aline C Simon; Maria Angeles Ortiz Bazan; Mairi L Kilkenny; David Wirthensohn; Mel Wightman; Dijana Matak-Vinkovíc; Luca Pellegrini; Karim Labib

doi:10.1016/j.molcel.2016.06.009

Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase

Mol Cell. 2016 Aug 4;63(3):385-96. doi: 10.1016/j.molcel.2016.06.009. Epub 2016 Jul 7.

Authors

Fabrizio Villa¹, Aline C Simon², Maria Angeles Ortiz Bazan¹, Mairi L Kilkenny², David Wirthensohn³, Mel Wightman¹, Dijana Matak-Vinkovíc³, Luca Pellegrini⁴, Karim Labib⁵

Affiliations

¹ MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
² Department of Biochemistry, University of Cambridge, 80, Tennis Court Road, Cambridge CB2 1GA, UK.
³ Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
⁴ Department of Biochemistry, University of Cambridge, 80, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address: lp212@cam.ac.uk.
⁵ MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Electronic address: kpmlabib@dundee.ac.uk.

Abstract

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation.

MeSH terms

Binding Sites
Chromosomes, Fungal / enzymology*
Chromosomes, Fungal / genetics
DNA Helicases / genetics
DNA Helicases / metabolism*
DNA Polymerase I / metabolism
DNA, Fungal / biosynthesis*
DNA, Fungal / genetics
DNA, Ribosomal / biosynthesis*
DNA, Ribosomal / genetics
DNA-Binding Proteins / genetics
DNA-Binding Proteins / metabolism*
Gene Dosage
Intracellular Signaling Peptides and Proteins / genetics
Intracellular Signaling Peptides and Proteins / metabolism*
Models, Molecular
Multiprotein Complexes
Mutation
Protein Binding
Protein Interaction Domains and Motifs
S Phase*
Saccharomyces cerevisiae / enzymology*
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae / growth & development
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism*
Structure-Activity Relationship

Substances

CTF4 protein, S cerevisiae
DNA, Fungal
DNA, Ribosomal
DNA-Binding Proteins
Intracellular Signaling Peptides and Proteins
Multiprotein Complexes
Saccharomyces cerevisiae Proteins
TOF2 protein, S cerevisiae
DNA Polymerase I
DNA Helicases
DNA2 protein, S cerevisiae

Abstract

MeSH terms

Substances

Grants and funding