Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells.
The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.
Cotta-Ramusino
Cecilia
C
Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20141 Milano, Italy.
Fachinetti
Daniele
D
Lucca
Chiara
C
Doksani
Ylli
Y
Lopes
Massimo
M
Sogo
José
J
Foiani
Marco
M
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004261:DNA Replication / Q000187:drug effects; D004271:DNA, Fungal / Q000096:biosynthesis / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006918:Hydroxyurea / Q000494:pharmacology; D008854:Microscopy, Electron; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D006918:Hydroxyurea