Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation.
DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600-base-pair (bp) region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M-G1 transition. top1 top2 double mutants exhibit fork block and processing and phosphorylation of Rad53 and gamma H2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation, and chromosome breakage during segregation.
Bermejo
Rodrigo
R
Fondazione Italiana per la Ricerca sul Cancro (FIRC), Institute of Molecular Oncology Foundation (IFOM-IEO Campus) and DSBB-Università degli Studi di Milano, 20139 Milan, Italy.
Doksani
Ylli
Y
Capra
Thelma
T
Katou
Yuki-Mori
YM
Tanaka
Hirokazu
H
Shirahige
Katsuhiko
K
Foiani
Marco
M
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2; D015825:Chromosomes, Fungal / Q000235:genetics / Q000378:metabolism; D016384:Consensus Sequence; D004249:DNA Damage; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II