title: Exo1 phosphorylation status controls the hydroxyurea sensitivity of cells lacking the Pol32 subunit of DNA polymerases delta and zeta.
abstract: Exo1 belongs to the Rad2 family of structure-specific nucleases and possesses 5'-3' exonuclease activity on double-stranded DNA substrates. Exo1 interacts physically with the DNA mismatch repair (MMR) proteins Msh2 and Mlh1 and is involved in the excision of the mispaired nucleotide. Independent of its role in MMR, Exo1 contributes to long-range resection of DNA double-strand break (DSB) ends to facilitate their repair by homologous recombination (HR), and was recently identified as a component of error-free DNA damage tolerance pathways. Here, we show that Exo1 activity increases the hydroxyurea sensitivity of cells lacking Pol32, a subunit of DNA polymerases δ and ζ. Both, phospho-mimicking and dephospho-mimicking exo1 mutants act as hypermorphs, as evidenced by an increase in HU sensitivity of pol32Δ cells, suggesting that they are trapped in an active form and that phosphorylation of Exo1 at residues S372, S567, S587, S692 is necessary, but insufficient, for the accurate regulation of Exo1 activity at stalled replication forks. In contrast, neither phosphorylation status is important for Exo1's role in MMR or in the suppression of genome instability in cells lacking Sgs1 helicase. This ability of an EXO1 deletion to suppress the HU hypersensitivity of pol32Δ cells is in contrast to the negative genetic interaction between deletions of EXO1 and POL32 in MMS-treated cells as well as the role of EXO1 in DNA-damage treated rad53 and mec1 mutants.
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, United States; Graduate Program in Cell and Molecular Biology, University of South Florida, Tampa, FL 33620, United States.
authors.forename: Lillian
authors.initials: L
authors.lastname: Doerfler
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, United States; Cancer Biology and Evolution Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, United States. Electronic address: kschmidt@usf.edu.
authors.forename: Kristina H
authors.initials: KH
authors.lastname: Schmidt
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D053843:DNA Mismatch Repair / Q000235:genetics; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D006918:Hydroxyurea / Q000494:pharmacology*; D051718:MutS Homolog 2 Protein / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D026923:Amino Acid Transport Systems, Basic; C502212:CAN1 protein, S cerevisiae; C522758:Pol32 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C090472:SGS1 protein, S cerevisiae; C489751:MSH2 protein, S cerevisiae; D051718:MutS Homolog 2 Protein; D053484:RecQ Helicases; D006918:Hydroxyurea