Rachel Kram

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 and DLD-1 is a colorectal adenocarcinoma-derived cell line. RAD18 had previously been implicated in DNA recombination. To characterize the RAD18- null cell lines, a series of recombination assays were 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 as a readout for the recombination frequency. DLD-1 RAD18-null cells did not prove to be hyper-recombinogenic. To assess the role of RAD18 in chromosomal radial formation in cancer cells, we examined the frequency of radial formations during metaphase before sister chromatids are separated. Radials are abnormal chromosomal structures that form as a result of DNA interstrand crosslinks (ICL), a form of DNA damage. Radials are a hallmark of Fanconi Anemia (FA) but the mechanisms behind radial formation are currently poorly understood. These experiments are currently ongoing. Our current studies are aimed at better understanding the underlying mechanism of chromosomal radial formations in human cancer cells and how RAD18 signaling regulates this process.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 and DLD-1 is a colorectal adenocarcinoma-derived cell line. RAD18 had previously been implicated in DNA recombination. To characterize the RAD18- null cell lines, a series of recombination assays were 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 as a readout for the recombination frequency. DLD-1 RAD18-null cells did not prove to be hyper-recombinogenic. To assess the role of RAD18 in chromosomal radial formation in cancer cells, we examined the frequency of radial formations during metaphase before sister chromatids are separated. Radials are abnormal chromosomal structures that form as a result of DNA interstrand crosslinks (ICL), a form of DNA damage. Radials are a hallmark of Fanconi Anemia (FA) but the mechanisms behind radial formation are currently poorly understood. These experiments are currently ongoing. Our current studies are aimed at better understanding the underlying mechanism of chromosomal radial formations in human cancer cells and how RAD18 signaling regulates this process.