Cancer is one of the leading causes of death in the United States. Genetic instability is an enabling cancer hallmark that facilitates the accumulation of progressively more malignant cancer phenotypes. To prevent genetic instability, cells have developed multiple evolutionarily conserved DNA damage repair and DNA damage response pathways that protect both the nuclear and mitochondrial genomes. One major cause of DNA damage is oxidative DNA damage, which results from reactive oxygen species (ROS). The major repair pathway for oxidative DNA damage is the Base Excision Repair (BER) pathway which is conserved from yeast to man. Although the BER pathway plays a major role in protecting both the nuclear and mitochondrial genomes, how this pathway is regulated is not known. Our recent studies have addressed this gap in knowledge by focusing on S. cerevisiae Ntg1, an evolutionarily conserved BER protein that recognizes and excises oxidized base lesions. The human Ntg1 counterpart, Nthl1, has altered function/localization that is linked to both colon and gastric cancers highlighting the importance of Nthl1 in preventing cancer and of defining mechanisms that regulate the BER pathway. Our work reveals that both Ntg1 and Nthl1 can be targeted to the nucleus or mitochondria in response to organelle-specific DNA damage. Consistent with a model where posttranslational modification(s) of BER proteins contribute to relocalization and/or regulation, our preliminary studies in budding yeast reveal that the nuclear pool of Ntg1 is modified by the Small Ubiquitin-like Modifier (SUMO) following nuclear oxidative damage. Importantly, preliminary data show that human Nthl1 can also be SUMO modified. To assess the functional importance of SUMO modification of these BER proteins, we have mapped all the SUMO modification sites on Ntg1 (K20,38,376,388,396) and created a non-sumoylatable (K->R) ntg1 variant (ntg1?SUMO). Although recombinant ntg1?SUMO retains catalytic activity in an in vitro assay, cells expressing ntg1?SUMO as the sole copy of Ntg1 exhibit a defect in the DNA damage response failing to arrest the cell cycle in response to DNA damage. Based on this preliminary data, I hypothesize that DNA damage-triggered SUMO modification of key BER proteins is required to orchestrate a proper DNA damage response. To test my hypothesis, the ntg1?SUMO mutant will be employed for the following Specific Aims: 1) to assess how SUMO modification impacts the repair capacity of Ntg1; 2) to explore connections between DNA damage-induced SUMO modification of Ntg1 and the DNA damage response pathway; and 3) to identify SUMO-dependent interactions with Ntg1 that could coordinate Ntg1 function with other cellular pathways. The proposed experiments seek to understand the role that Ntg1 plays in the DNA damage checkpoint and identify the sumoylation-dependent interactions required for this function. My long term goals are to understand the communication between BER and other DNA damage response pathways to provide insight into the mechanisms that regulate BER and possibly suggest new therapeutic targets for cancer.
A major contributing factor to the development and progression of cancer is the accumulation of mutations in the genetic material. Evolutionarily conserved DNA repair pathways eliminate damage to the DNA to protect the genome from these mutations. Understanding how DNA repair pathways are regulated and communicate with other cellular protection pathways to maintain normal cell growth and prevent cancer- causing events could lead to novel approaches for treating cancer.
| Swartzlander, Daniel B; McPherson, Annie J; Powers, Harry R et al. (2016) Identification of SUMO modification sites in the base excision repair protein, Ntg1. DNA Repair (Amst) 48:51-62 |
