The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

Abstract

In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with "repairable" DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a "last-resort" mechanism for cell survival.KEYWORDS Replication Protein A; DNA damage; checkpoint adaptation; phosphorylation; genetic interaction C ELLS encounter environmental stress on a continual basis and have evolved mechanisms to monitor the integrity of the genome and prevent temporary DNA lesions from becoming permanent DNA mutations. A central factor in genome monitoring is the protein complex Replication Protein A (RPA). The canonical RPA complex is composed of three subunits named RPA1, RPA2, and RPA3, also often referred to by their apparent molecular weights as RPA70, RPA32, and RPA14, respectively (Wold 1997;Iftode et al. 1999;Fanning et al. 2006;Zou et al. 2006;Oakley and Patrick 2010). Originally identified as a protein complex essential for in vitro SV40 DNA replication (Wold and Kelly 1988;Wold et al. 1989;Weinberg et al. 1990), this complex is also essential for DNA repair/ recombination (Longhese et al. 1994;Firmenich et al. 1995;Sung 1997;Umezu et al. 1998) and has roles in cell-cycle regulation (Longhese et al. 1996;Lee et al. 1998;Anantha et al. 2008;Anantha and Borowiec 2009). This is consistent with the major biochemical function of RPA, which is highaffinity binding to single-strand DNA (ssDNA), an intermediate of replication, repair/recombination, and substrate for checkpoint activation (Smith et al. 2010;Flynn and Zou 2010;Mimitou and Symington 2011;Ashton et al. 2013). In addition to acting as a "sensor" of DNA damag...

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“…The ssDNA that is generated is immediately coated by the ssDNA binding protein complex replication protein A (RPA). RPA-coated ssDNA protects the DSB ends from further degradation and signals to the cell the presence of unrepaired DNA damage (Ghospurkar et al 2015; Manfrini et al 2015; Sung and Klein 2006; Wold 1997). …”

The Multiple Roles of Ubiquitylation in Regulating Challenged DNA Replication

“…Replication protein A (RPA) complex is composed of three subunits (Rfa1/RPA70, Rfa2/RP32 and Rfa3/RPA14) and is phosphorylated by checkpoint kinases (Vassin et al 2009;Ghospurkar et al 2015). It has been recently described that all RPA subunits are ubiquitylated in response to replication fork stalling induced by agents causing dNTP depletion or DNA damage (Elia et al 2015).…”