Nucleolytic processing of aberrant replication intermediates by an Exo1-Dna2-Sae2 axis counteracts fork collapse-driven chromosome instability

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.Spanish Ministry of Science and Innovation [RYC-2010-07131, BFU2011-24909 to R.B.]; Spanish Ministry of Economy and Competitiveness [BFU2014-52529-R to R.B.]; European Community's 7th Framework Programme [n° 293770 to R.B.]; Spanish National Research Council (CSIC) [PII-201410I001 to R.B.]; Italian Foundation for Cancer Research (F.I.R.C.) (to A.C.); Education Council of the Castilla y León regional government (to C.F.) Spanish Formación del Personal Investigador (FPI) programme (to G.P., S.V.). Funding for open access charge: Spanish Ministry of Economy and Competitiveness; European Union.EUR 2,000 APC fee funded by the EC FP7 Post-Grant Open Access PilotPeer reviewe