Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

CELLS 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,andRPA3, also often referred to by their apparent molecular weights as RPA70, RPA32,andRPA14, 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 damage through its ability to bind to ssDNA, RPA is also post-translationally modified in response to DNA damage. Identified post-translational modifications of RPA include acetylation (Choudhary et al. 2009), sumoylation (Burgess et al. 2007; Dou et al. 2010), and phosphorylation (Din et al. 1990; Dutta et al. 1991; Liu et al. 1995, 2005, 2012; Henricksen et al. 1996; Brush et al. 1996; Brush and Kelly 2000; Kim and Brill 2003; Vassin et al. 2004; Olson et al. 2006; Anantha et al. 2007, 2008; Lee et al. 2010; Shi et al. 2010; Wang et al. 2013). Most studies of RPA post-translational modifications have focused on hyperphosphorylation of the 40-amino-acid (aa) N-terminal region of human RPA2 in response to DNA damage. The use of "extensive" phospho-mutants (i.e., those in which all serines/ threonines in the region are changed to aspartic acids to mimic phosphorylation or alanines to prevent phosphorylation) indicates that mimicking a hyperphosphorylated state results in the inability to detect RPA2 foci at replication centers in otherwise unstressed human cells (Vassin et al. 2004). This suggests that in response to DNA damage, phosphorylated human RPA is recruited away from replication centers to perform functions in DNA repair. Mutagenesis studies have also indicated that phosphorylation of the human RPA2 N-terminus (NT) is important for halting the cell cycle during replicative stress (Olson et al. 2006), for progression into mitosis (Oakley et al. 2003; Anantha et al. 2008; Anantha and Borowiec 2009), and for differential protein interactions with some DNA-damage response proteins (Oakley et al. 2003, 2009; Patrick et al. 2005; Wu et al. 2005).

Within the human RPA2 NT are nine serine/threonine (S/T) residues that are targets for phosphorylation (Iftode et al. 1999; Anantha et al. 2007; Liu et al. 2012). The combination of various RPA2 phospho-mutants and the generation of phospho-specific human RPA2 antibodies have advanced this area of research by allowing for the examination of phosphorylation at each individual target residue. The sites in the human RPA2 NT appear to be differentially phosphorylated in response to various types of DNA damage (Liu et al. 2012), likely due to different checkpoint kinases (e.g., ATR, ATM, and DNA-PK) having different preferential targets within the RPA2 NT (Brush et al. 1996; Olson et al. 2006; Cruet-Hennequart et al. 2008; Vassin et al. 2009; Liaw et al. 2011; Liu et al. 2012). Also, sequential phosphorylation of the human RPA2 NT has been reported, indicating a dependence on phosphorylation of one site to promote phosphorylation of another (Anantha et al. 2007; Liu et al. …

Search by... Author
Show... All Results Primary Sources Peer-reviewed

Oops!

An unknown error has occurred. Please click the button below to reload the page. If the problem persists, please try again in a little while.