Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.
Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.
Sogo
José M
JM
Institute of Cell Biology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
Lopes
Massimo
M
Foiani
Marco
M
D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D011564:Furocoumarins / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D008854:Microscopy, Electron; D009154:Mutation; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D011564:Furocoumarins; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D006918:Hydroxyurea