Dueling Proteins Control Replication Fork Stability
A variety of cell stressors may stall the process of DNA replication, and failure to resolve the problem and resume normal progression of the replication fork may lead to DNA damage and/or even cell death. Stalling of the replication fork results in exposure of single stranded DNA (ssDNA), so ssDNA binding proteins are usually the first to respond, both to protect the DNA and trigger the replication stress response. One such protein is RAD51, which promotes the process of fork reversal in which a portion of the unwound DNA template reanneals, and the newly synthesized strands anneal with each other to form a four-branched structure. Fork reversal may serve as a first step towards resolution of replication stress; however, if prolonged, reversal may lead to a broken or collapsed fork. Opposing the function of RAD51 is RADX, a protein recently discovered in the laboratory of Vanderbilt Basic Sciences investigator David Cortez. Now, the Cortez lab reports new studies showing that suppression of RAD51 function via multiple mechanisms in U2OS osteosarcoma cells leads to instability of reversed forks accompanied by degradation of ssDNA. Fork stabilization is restored by reducing the expression of RADX, suggesting that a balance between the two proteins is critical to fork integrity. Consistently, overexpression of RADX resulted in excessive ssDNA degradation that was blocked by suppression of fork reversal-promoting proteins that function in the absence of RAD51. Further studies demonstrated that partial knockdown of RAD51 leads to fork destabilization whereas complete knockdown stabilizes the fork by preventing the fork reversal process. This led to the hypothesis that low concentrations of RAD51 lead to fork reversal whereas high concentrations protect the reversed fork. Overexpression of RADX protects forks destabilized by partial RAD51 knockdown by opposing the remaining RAD51 activity. Competitive binding studies demonstrated that >10,000-fold higher concentrations of RAD51 are required to displace RADX on ssDNA, but cells contain higher concentrations of RAD51 than RADX, and multiple proteins facilitate and stabilize RAD51 binding to compensate for the difference in affinity between the two proteins. The investigators conclude that RADX functions to fine-tune the role of RAD51 in fork reversal; however, further work is needed to fully understand how the two proteins interact to protect the fork both during normal replication and at times of stress. The work is published in the journal Cell Reports[K. P. Bhat, et al., (2018) Cell Rep., 24, 538].