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Using budding yeast to study properties and function of the human Replication Protein A2 N‐terminus in response to specific‐types of DNA damage
Author(s) -
Haring Stuart J.,
Senger Barbara L.,
Wilson Timothy M.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.573.4
Subject(s) - phosphorylation , replication protein a , biology , microbiology and biotechnology , origin recognition complex , eukaryotic dna replication , dna replication , biochemistry , dna , gene , dna binding protein , transcription factor
Background Replication Protein A (RPA) is a heterotrimeric complex that serves critical roles in eukaryotic genome duplication and maintenance. In human cells, the N‐terminus (NT) of the 32‐kDa subunit of RPA (Rpa2) is hyper‐phosphorylated by multiple kinases in response to DNA damage. We demonstrated previously in the budding yeast Saccharomyces cerevisiae that phosphorylation of the yeast Rpa2 NT does not occur under commonly assayed genotoxic conditions, yet this domain is vital for the damage response. Using a chimeric yeast Rpa2 protein containing the human Rpa2 N‐terminus, we have demonstrated that this human domain is phosphorylated in yeast cells a manner nearly indistinguishable from that in human cells. Study Objective We wish to determine properties of human Rpa2 NT phosphorylation, including kinases required, sites involved and their interdependence, and what conditions trigger this phosphorylation to identify whether properties of Rpa2 NT phosphorylation are conserved in yeast and human cells. Methods We utilized chimeric human‐yeast Rpa2 proteins (with WT and mutant NT), yeast Rpa2 antibodies, and human Rpa2 phospho‐specific antibodies to examine the potential for the human Rpa2 NT to be phosphorylated in response to varying DNA damaging agents. Results and Conclusions We determined that the same sites in the human Rpa2 NT are phosphorylated on the chimeric protein in yeast, and that these show similar dependence on DNA damage induction. Unlike that reported in human cells, phosphorylation of one site does not depend on phosphorylation of another site within the domain ( i.e. , no sequential/primed phosphorylation). We demonstrated that phosphorylation of the human Rpa2 NT in yeast only appears to occur when cells are treated with agents that produce double‐strand breaks ( e.g. , HO endonuclease, phleomycin, camptothecin) but not when replication is inhibited by hydroxyurea. Finally, we have shown that Mec1 (yeast ATR), but surprisingly not Tel1 (yeast ATM), is necessary to phosphorylate T21 and S33 (both containing TQ or SQ) on the human Rpa2 NT. This is in contrast to human cells where ATR has only been reported to phosphorylate S33, not T21. We conclude that yeast provides a simple system to identify kinases involved, conditions that trigger human Rpa2 phosphorylation, and its relation to other properties of damage responses. This also provides the opportunity to study Rpa2 NT modification in two different systems to gain an understanding of conserved properties and to potentially study the significance of differences in the damage response between the two systems. Support or Funding Information This research was supported by NSF‐CAREER 1253723 to SJH.