Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis that are observed in abnormally dividing cells. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to copy the genomic template and leads to the arrest of the replication machinery and gaps in the newly replicated DNA. The overall objective of this research is to identify the cellular mechanism(s) by which lesions encountered during replication are processed and repaired in vivo. This project utilizes two previously established cellular assays to monitor the molecular events that occur at replication forks arrested by DNA damage in vivo. This approach will identify the genetic requirements and conditions that determine when DNA repair or translesion synthesis functions at arrested replication forks to restore DNA synthesis. A second approach will characterize the genes and mechanisms that operate to process and repair lesions in the gapped substrates that are observed to arise in the newly replicated DNA of UV-irradiated cells. Through an understanding of how faithful replication is maintained in the presence of DNA damage, the conditions and events that can lead to mutagenesis, genomic rearrangements, or cell lethality will be elucidated. In addition, this project will be accomplished through a process that strengthens the undergraduate curriculum, creates a new molecular genetics laboratory for students, and increases student participation in research at this university. This project will significantly increase the number of potential young scientists that participate in research and increase the quality of science that students are exposed to in this region of the country.
Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis that are observed in cancer cells. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to copy the genomic template. Significant advances have been made in identifying gene products that impair survival after DNA damage. However, the cellular mechanism(s) by which DNA damage is processed during replication in vivo remains relatively uncharacterized. Following UV-induced DNA damage in Escherichia coli, replication can either arrest at the lesion site, or skip over the damage, generating a gap. When replication forks are arrested, they are processed and maintained by several proteins belonging to the recF pathway prior to recovery. The processing involves a transient reversal of the replication fork that has been postulated to allow repair enzymes or translesion DNA polymerases to gain access to the blocking lesion and effect repair. This research has identified several of the genetic requirements and conditions that determine when repair and translesion synthesis occur at arrested replication forks to restore replication. We have characterized the role that several genes, known to be impaired for survival after DNA damage, have in processing and restoring replication following damage. In a collaborative the structure of the RecF protein, which is central to this process, was solved and a structure-functional analysis of the protein was initiated. The work has also demonstrated that other gene products, which were originally postulated to be involved in restoring replication based on biochemical studies, do not affect the ability of cells to resume DNA synthesis after DNA damage in vivo. The results have advanced our understanding of how faithful replication resumes when it is blocked by DNA damage and defined conditions and events that can lead to mutagenesis, genomic rearrangements, and lethality in the presence of DNA damage. In addition, this research has been carried out at Portland State University, an urban institution that enrolls over 20,000 undergraduate and 3,800 graduate students, making it Oregon’s largest university. Over 60% of the state’s residents live within commuting distance of the campus, and PSU has grown to become Oregon’s primary vehicle for meeting the state’s pre-professional and graduate education needs. PSU now has the largest enrollment of MS and PhD students in the state and sends more undergraduates to nursing, dental, or medical school than any other Oregon university. We have been able to effectively target our outreach programs and coursework with high school and community colleges, resulting in an unusually large proportion of new and first generation professionals among our PSU graduates. The Biology Department has been integral to the growth of PSU, seeing our student enrollment increase by 65% in the last 5 years with growth projected to continue at a similar rate in the near future. In 2004, we applied for and were awarded a PhD program for Biology by the State of Oregon, highlighting our growth in research strength in recent years. Our department has pursued a consistent vision that has transformed the biology experience for undergraduates by focusing on 1) developing a research active faculty committed to undergraduate education, 2) providing undergraduate laboratory experiences that involve the latest equipment and software, 3) ensuring that our best and most committed students experience research as undergraduates, and 4) developing effective and successful outreach programs to serve our community colleges and Oregon’s K-12 teachers, which ultimately enhances the quality and performance of the students in our pipeline when they enter our program. This research has provided training and research experiences for 7 graduate students, 12 undergraduates and 2 teachers from undergraduate colleges. I have taught three courses per year during the course of this work, Introductory Genetics ~170 students/quarter, Microbial Genetics ~ 20 students/quarter, and Graduate Grant Writing ~25 students/quarter. Finally, this grant has allowed me to contribute to the development and growth of our program in several ways, including serving as Chair of our new Ph.D. program in Biology, serving as Coordinator for our undergraduate labs and graduate teaching assistantships, and in development of a weekly Biology Seminar series that has served both our undergraduate and graduate populations.
