Abstract
Problems during DNA replication underlie genomic instability and drive malignant transformation. The DNA damage checkpoint stabilizes stalled replication forks thus counteracting aberrant fork transitions, DNA breaks and chromosomal rearrangements. We analyzed fork processing in checkpoint deficient cells by coupling psoralen crosslinking with replication intermediate two-dimensional gel analysis. This revealed a novel role for Exo1 nuclease in resecting reversed replication fork structures and counteracting the accumulation of aberrant intermediates resembling fork cleavage products. Genetic analyses demonstrated a functional interplay of Exo1 with Mus81, Dna2 and Sae2 nucleases in promoting cell survival following replication stress, suggestive of concerted nucleolytic processing of stalled forks. While Mus81 and other Structure Specific Endonucleases do not contribute to obvious collapsed fork transitions, Dna2 promotes reversed fork resection likely by facilitating Exo1 access to nascent strands. Instead, Sae2 cooperates with Exo1 in counteracting putative fork cleavage events linked to double strand breaks formation and increased gross chromosomal rearrangement rates. Our data indicate that in checkpoint deficient cells diverse nuclease activities interface to eliminate aberrant replication intermediates and prevent chromosome instability.
Highlights
Preventing errors during DNA replication is essential to guarantee the correct transmission of genetic information
The data here presented establish a direct link between stalled fork nucleolytic processing, DSB formation and chromosomal rearrangements
In checkpoint deficient cells, concerted fork processing prevents the accumulation of aberrant intermediates, some of which likely prime DSBs and chromosome instability
Summary
Preventing errors during DNA replication is essential to guarantee the correct transmission of genetic information. The DNA damage checkpoint is a key mechanism that eukaryotic cells have evolved to protect genome integrity during replication [6,7]. The checkpoint response is coordinated by the central protein kinases Mec1/ATR and Rad53/CHK2, which ensure the stabilization and timely restart of stalled forks, essential in turn for viability and genome integrity maintenance in cells experiencing replication stress [8,9]. Stalled forks accumulate Replication Protein A (RPA)-covered single stranded DNA (ssDNA) that recruits Mec1/ATR, subsequently triggering Rad53/CHK2 activation [11,12]. Rad plays a key role in preserving both the structural integrity of replication intermediates and the proficiency for DNA synthesis of stalled forks [6,15,16,17,18]. The DNA damage checkpoint preserves genome integrity by modulating chromosome architecture to relieve topological stress [19] and by inhibiting late-origin firing [20]
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