| Polymerase stalling during DNA replication results in uncoupling of DNA polymerase and the replicative helicase and the consequent generation of single-stranded DNA (ssDNA), which is the primary signal for the ATR-mediated S phase checkpoint. Although polymerase stalling is not necessarily associated with DNA damage, double-strand breaks (DSBs) do accumulate over time, especially when cells resume DNA replication. These DSBs are repaired predominantly by a homologous recombination (HR) process. The key enzyme that mediates HR is the RAD51 recombinase, which catalyzes homology-dependent strand invasion and D-loop formation. In order to catalyze strand invasion, RAD51 must be loaded onto ssDNA by mediators, such as RAD52, that are charged with substituting RAD51 for ssDNA-binding replication protein A (RPA). Recent studies have shown that the BLM helicase acts during these initiating steps of HR, with the capacity to facilitate or deny access of RAD51 to ssDNA. BLM is modified by the small ubiquitin-related modifiers (SUMOs), and I have hypothesized that BLM SUMOylation acts as a switch to regulate its pro- and anti-recombinogenic functions at stalled replication forks.;In order to investigate the mechanism by which SUMOylation regulates BLM function, I stably expressed in BLM-null cells either normal BLM (BLM+) or SUMO-mutant BLM (SM-BLM), which contains mutations at two SUMO-acceptor sites K317 and K331, and treated the cells with hydroxyurea (HU), which stalls DNA polymerases by nucleotide deprivation. Although SM-BLM cells accumulated more DSBs than BLM+ cells, SM-BLM cells exhibited a defect in induction of HR repair as evidenced by lower levels of HU-induced sister-chromatid exchanges. Consistent with this defect, in HU-treated SM-BLM cells, RAD51 failed to co-localize normally with BLM and gamma-H2AX -- a marker for DNA damage. In vitro, RAD51 bound SUMOylated BLM more efficiently than unSUMOylated BLM, indicating that RAD51 contains a SUMO binding site and suggesting that BLM SUMOylation could help recruit RAD51 to the repair site through SUMO -- SUMO binding-site interactions.;To investigate the mechanism by which BLM SUMOylation influences RAD51 localization, I compared RAD52 and RPA localization at stalled forks in SM-BLM and BLM+ cells. While HU-treated BLM+ cells exhibited a 1.6 fold increase of RAD52 foci per cell over untreated BLM+ cells, HU treatment did not induce a significant increase of RAD52 foci in SM-BLM cells. On the other hand, SM-BLM cells exhibited dramatically increased levels of RPA foci compared to BLM+ cells in both untreated and HU-treated conditions. In addition to an excess in focal concentrations of RPA in SM-BLM cells, there are higher levels of HU-induced chromatin-bound RPA and increased HU-induced BLM-RPA interaction, as evidenced by immunoprecipitation of endogenous proteins. These striking differences in RPA accumulation at stalled forks in SM-BLM cells suggest that BLM SUMOylation directly regulates the amount of ssDNA that accumulates during polymerase stalling, supporting the hypotheses that BLM SUMOylation could either inhibit BLM DNA helicase activity to suppress the formation of aberrant ssDNA at stalled forks, or alternatively it could participate in a process that re-anneals ssDNA at stalled forks. |
