Rachel Kram

Authors: Rachel E. Kram, Colette B. Rogers, Anja-Katrin Bielinsky

The RAD proteins were originally identified in yeast as being radiation sensitive (RAD). Human homologues of many of these proteins have since been identified. RAD18 is an E3 ubiquitin ligase that ubiquitinates a protein known as proliferating cell nuclear antigen (PCNA) in response to replication stress and RAD18 is frequently upregulated in cancer. The goal of this project was to gain better insight into the mechanism of how RAD18 promotes cancer cell survival. RAD18 was knocked out in two human cell lines, U2OS and DLD-1. U2OS is an osteosarcoma cell line, that uses a process called Alternative Lengthening of Telomeres (ALT) for telomere maintenance. This is a telomerase independent mechanism to prevent telomere shortening during DNA replication. About 90% of human cancers are telomerase positive and 10% are ALT positive. DLD-1, a colorectal adenocarcinoma-derived cell line, is dependent on the activity of telomerase to maintain its telomeres. In order to characterize the RAD-18 null cell lines, a series of recombination assays was performed. RAD18 mutants accumulate single stranded gaps in their genomes and rely on homologous recombination to repair them. A sister chromatid exchange (SCE) assay was used to assess homologous recombination. In SCE, the crossover is a readout for the recombination frequency. A separate assay was used to determine the rate of SCE’s in the telomeres of the two cell lines. These experiments are currently ongoing. Our study addresses the consequences of inhibiting RAD18-dependent signaling pathways in telomerase+ and ALT+ cancers.