Loss of function of the WRN protein causes the genetic disorder Werner Syndrome that is characterized by increased cancer and premature aging. WRN belongs to the RecQ helicase family that plays key roles in preventing genome instability. In response to treatment with genotoxins, WRN is subject to post-translational modification. The relationship of post-translational modification of WRN with its function in DNA metabolism is unknown. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in DNA replication. Consistent with this notion, WS cells are sensitive to DNA replication inhibitors and DNA damaging agents that tend to block replication fork progression. The cells exhibit an extended S phase, as well as defects in normal bi-directional progression of replication forks diverging from the majority of replication origins. To elucidate the relationship between post-translational modifications of WRN with its function in DNA metabolism, here the acetylation of WRN was studied. In our studies, we provide evidence that WRN acetylation is a dynamic process that strongly correlates to blockage of replication by persistent DNA damage. We also determined the effect of WRN acetylation on its specificity and enzymatic functions. In addition, our studies reveal how agents that block replication regulate the nature of WRN interactions with RPA, a factor known to bind to single-stranded DNA generated at blocked replication forks. Our results demonstrated that WRN and RPA form a stable direct association under normal physiological conditions and treatments that block replication fork progression increase their association, further supporting the idea that WRN is involved in DNA replication through its action at blocked or stalled replication forks. Thus, these studies point to both 1) an important role for acetylation of WRN and 2) its interaction with RPA in the putative function of WRN in response to blocked replication. Overall, our results impact knowledge regarding the relationship between DNA damage, genome instability and the development and progression of aging and cancer.

All human chromosomes end with protective structures called telomeres, which consist of thousands of double-stranded TTAGGG repeats and end in a 3’ guanine-rich overhang. These structures shorten normally during each round of replication, and extremely short telomeres along with telomere dysfunction are thought to contribute to the development of aging and cancer. Although many proteins have roles in telomere maintenance, WRN, which is a 3’ to 5’ helicase that is deficient in the premature aging disorder Werner’s syndrome, has been proposed to play multiple roles at telomeres. In this study, I focus on the effect of telomeric sequences and/or structures formed during DNA replication or recombination and how WRN functions at these sites. This study suggests that WRN may promote proper replication of telomeres by accurately aligning telomeric sequences during replication fork regression, potentially the first step in responding to a blockage, such as DNA damage. However, even in the presence of WRN, replication of telomeric sequences is difficult, possibly due to the ability of G-rich sequences to form secondary structures such as G-quadruplexes. I demonstrate that the translesion polymerase pol η, as well as a variety of other polymerases, is unable to synthesize past an intramolecular G-quadruplex formed from telomeric sequence on the template strand. Furthermore, in physiological salt concentrations, WRN favors binding and unwinding a structure that mimics a strand invasion intermediate over other similar structures especially when it possesses G-telomeric sequence. In addition, WRN promotes unwinding of these structures in a direction that would promote additional annealing and strand invasion, supporting a role for WRN in promoting telomeric recombination and formation of a T-loop, a proposed protective structure specific to telomeres. Overall, the data suggest that telomeres may pose problems in replication due to the G-rich, repeating nature of the structures, while WRN may aid in promoting proper replication at these and other replication blocks. Furthermore, WRN may play a role in promoting additional formation of T-loops and other telomeric recombination, thus supporting the relationship of WRN, telomere maintenance, and potentially development of certain aging characteristics.