title: Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing.
abstract: The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.
authors.affiliation: Istituto FIRC di Oncologia Molecolare, Via Adamello 16, Milano 20141, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Lucca
authors.forename: Fabio
authors.initials: F
authors.lastname: Vanoli
authors.forename: Cecilia
authors.initials: C
authors.lastname: Cotta-Ramusino
authors.forename: Achille
authors.initials: A
authors.lastname: Pellicioli
authors.forename: Giordano
authors.initials: G
authors.lastname: Liberi
authors.forename: James
authors.initials: J
authors.lastname: Haber
authors.forename: Marco
authors.initials: M
authors.lastname: Foiani
mesh_terms: D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D002869:Chromosome Aberrations; D004261:DNA Replication / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D015321:Gene Rearrangement; D020022:Genetic Predisposition to Disease / Q000235:genetics; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D004259:DNA-Directed DNA Polymerase