Genomic instability is one of the enabling hallmarks of cancer and can arise by exposure to several factors: endogenous challenges such as stress to DNA replication forks or exogenous compounds such as exposure to ionizing radiation (IR) and other environmental mutagens. These insults can lead to the induction and propagation of mutations through the generation of multiple DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different repair pathways. One of these pathways is homologous recombination (HR), a relatively faithful DSB repair pathway that relies on the availability of a homologous template for DNA repair synthesis.

DNA strand exchange in HR is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. In human cells, RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While RAD54 translocates on double-stranded DNA and promotes DNA strand separation, RAD51AP1 enhances the assembly of the synaptic complex. However, if RAD54 and RAD51AP1 work independently or together in the HR reaction is unclear. Since functional loss of either RAD54 or RAD51AP1 each leads to a moderate defect in HR, we speculate that RAD54 and RAD51AP1 may act in parallel in the promotion of RAD51 activity. We hypothesize that simultaneous inactivation of both RAD54 and RAD51AP1 may lead to a synthetic phenotype in HR impairment.

The main focus of this study is to investigate the potential synergism between RAD51AP1 and RAD54 during late stages of the HR reaction to improve targeted cancer therapy. We are investigating if loss of RAD54 in human cells exacerbates the phenotype of RAD51AP1-deficiency and renders cells more sensitive to DNA damaging agents. We have generated RAD51AP1 knockout HeLa cells using CRISPR/Cas9 technology. In these cells, we have also inactivated the RAD54 gene using a very similar approach. Moreover, we have generated RAD54 knockout (KO) cells in a wild type RAD51AP1 background. All cell lines are tested for their response to chemotherapeutic agents in cell survival and DNA replication assays. Our first results show that the double KO cells are more sensitive to mitomycin C exposure than single KO cells, suggesting that RAD51AP1 and RAD54 function largely independently of each other in the HR reaction.

The results from our investigation are expected to lead to the potential development of new cancer drugs targeted at ancillary factors of RAD51 in late stages of the HR reaction.

Citation Format: Platon Selemenakis, David Maranon, Claudia Wiese. Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2569.