Regulation of Rad18 Via the Ataxia Telangiectasia and Rad3 Related (ATR) Kinase
AU Gurkar, C Vaziri. Boston University, Boston, MA
Background: Carcinogenic DNA-damaging agents induce DNA lesions termed bulky adducts. During DNA replication, DNA polymerases encountering such adducts are stalled. Translesion synthesis (TLS) is a DNA repair mechanism by which, specialized DNA polymerases are recruited to stalled replication forks and perform DNA synthesis across damaged sites. Recruitment of TLS polymerases to stalled replication forks requires an E3 ubiquitin ligase RAD18. RAD18 functions by mono-ubiquitinating the polymerase processivity factor PCNA. PCNA interacts directly with TLS polymerases and acts as a polymerase switch, which enables cells to preserve replication forks that encounter DNA damage. Cells lacking RAD18 or TLS polymerases are highly sensitive to DNA damage-induced lethality. However, the mechanism(s) by which RAD18 senses DNA damage and gets recruited to the site of lesion are unclear. We and others have hypothesized that DNA damage-induced checkpoint signaling may promote TLS. Checkpoint kinases such as ataxia telangiectasia mutated and Rad3-related (ATR) have been previously reported to play a role in PCNA ubiquitination. A recent proteomic screen identified Ser 403 of RAD18 as a potential substrate for the checkpoint kinases ATM and ATR (Matsuoka et. al. Science 07). Therefore, ATM/ATR-mediated phosphorylation of RAD18 provides a potential mechanism for regulation of TLS by checkpoint signaling.
Design: To test our hypothesis that RAD18 is phosphorylated at S403 in response DNA damage we made a phosphorylation mutant (S403-A). Both ATM and ATR were identified as potential kinases for RAD18, therefore, we tested the hypothesis that checkpoint signaling regulates RAD18 in response damage. Also we tested whether S403 phosphorylation regulates RAD18-dependent TLS. This study provides new insights into the mechanisms by which integration of checkpoint signaling and TLS helps maintain genomic stability and prevent cancer.
Results: Our preliminary results suggest that Rad18 is phosphorylated basally at S403 site. Intrestingly, the phosphorylation at this site increases upon DNA damage. Our data also indicates that the phosphorylation at this site is required for successful cell cycle progression. Absence of phosphorylation (as tested using a S403 to A mutant) leads to accumulation of cells in the S phase. Failure to phosphorylate Rad18 at S403 also leads to upregulation of DNA damage markers such as P-Chk1 and P-H2Ax.
Conclusions: Rad18 S403 site seems to be phosphorylated upon DNA damage. This phosphorylation is required for cell cycle progression and viability upon DNA damage.
Tuesday, March 10, 2009 1:00 PM
Poster Session IV # 196, Tuesday Afternoon