DNA damage occurs in all organisms, both spontaneously and as a result of exposure to various environmental insults. Oxidative damage to DNA, which accounts for most of the naturally occurring instances of DNA damage, is repaired via the Base Excision Repair (BER) pathway. The enzymes that carry out BER have been extensively studied using naked DNA templates, but there is very little information on how these same enzymes gain access to their substrates in the nuclei of eukaryotic cells, where the DNA is packaged into chromatin. The fundamental subunit in chromatin is the nucleosome, which consists of DNA wrapped around a protein core. To elucidate mechanisms by which BER enzymes gain access to damaged DNA in chromatin, the PI and his collaborator established a model system in which nucleosomes, with defined DNA lesions at pre-determined sites, are assembled in vitro and incubated with selected, purified human or bacterial BER enzymes. This model system will be used first to determine if the efficiency of excision of damaged bases from nucleosomal DNA is influenced by the chemical nature of the damage itself or by possible nucleosome-imposed constraints to changes in DNA conformation that must occur during BER (e.g., base flipping). Second, preliminary studies indicate that certain human BER enzymes are more efficient than the equivalent bacterial enzymes at processing DNA damage in nucleosomes. Deletion mutants will be used to identify domains within the human enzymes that account for this elevated efficiency. Finally, oxidative damage to DNA sometimes occurs in clusters, and attempted BER of closely juxtaposed lesions can lead to double-strand DNA breaks that may be lethal or mutagenic. Model nucleosomes containing DNA damage clusters will be used to determine how the packaging of DNA in chromatin affects the frequency or pattern of BER-induced, double strand break formation.
BROADER IMPACTS: The research embodied in this project will provide new information on how BER enzymes are recruited to and act on DNA damage in chromatin, an important area of inquiry where, at present, very little is known. Because such damage must be repaired to ensure the survival of individual organisms and their capacity to propagate from one generation to the next, this information will be relevant to the many scientists who study DNA packaging, genome stability, or other metabolic transactions involving DNA. Results from this research project will be broadly disseminated through the research literature and presentations at national and international meetings. In addition, the PI and his collaborator deposit all data in the UVM BioDesktop repository, and this information is posted on the web as soon as the corresponding article is accepted for publication. This project also will enhance the scientific infrastructure at UVM by strengthening the ongoing scientific interactions between the PI, whose lab has expertise in chromatin and DNA replication, and Dr. Susan Wallace, whose lab has expertise in BER. Finally, this project will provide high quality training opportunities for undergraduate and graduate students. The multidisciplinary efforts in both laboratory groups encompass biochemical and molecular methods as well as genetic, bioinformatic and crystallographic approaches that collectively provide a stimulating environment for students and postdoctoral trainees. Both the PI and his collaborator have solid records of training these individuals. Both teach undergraduate and graduate courses in a Department that offers two undergraduate majors and provides students in these majors the opportunity to conduct laboratory research. The Department's graduate program is among the most highly regarded of the graduate programs in the biological sciences at the University. Special attention is paid to cultivating students from underrepresented backgrounds. Thus, this project will help strengthen our national scientific infrastructure.
Embedded in the DNA of every cell are instructions needed for normal growth and metabolism. Chemical or physical damage to DNA may alter or destroy this information, resulting in altered cell function or death. Key to survival, therefore, are the numerous enzymes that detect and repair damage in DNA. The challenge that such enzymes face is that DNA is packaged in chromatin, which severely limits its accessibility. Most of chromatin consists of tandemly arrayed nucleosomes, each of which consists of a discrete sized DNA segment, wrapped about a histone protein core. The overall goal of this Project was to identify molecular mechanisms that enable base excision repair (BER) factors to repair oxidatively damaged DNA bases in nucleosomes. Prior to initiating this project we had a set of observations that, collectively, suggested that intrinsic nucleosome dynamics facilitate the binding of BER factors to damaged bases that are normally inaccessible, because of their orientation relative to the underlying histone core. Spontaneous, partial unwrapping of DNA from the histone core transiently exposes these damaged bases, during which time they can be bound by BER factors. With NSF support, we developed and tested a kinetic model that relates the frequency and duration of these DNA unwrapping events to the rate of repair of lesions in nucleosomes. The manuscript reporting this work has been revised to address reviewer comments and is nearly ready for resubmission. With NSF support, we also investigated parameters that enable BER enzymes to discover oxidatively damaged DNA bases in a sea of undamaged DNA. This required that we measure the in vivo concentration of selected BER enzymes as well as their specific and non-specific DNA binding constants. The resulting measurements enabled us to predict that the DNA glycosylase hNTH1 binds DNA in a non-specific fashion, and then scans along DNA until it encounters a damaged base. This is probably not the case for a second DNA glycosylase, NEIL1, which may need to partner with other factors during the search for DNA damage. Finally, NSF support enabled us to investigate the hypothesis that attempted BER of clustered oxidative lesions in chromatin can produce potentially lethal double strand DNA breaks. We discovered that nucleosomes only partially protect DNA from double strand breaks. This result helps explain the origin of the ~50 double breaks that occur daily in each human cell. We currently are preparing a manuscript that will report these findings. In summary, with NSF support, we have generated novel findings that advance our knowledge of nucleosome dynamics, molecular mechanisms that facilitate BER, and mechanisms by which BER factors sometimes damage DNA, by generating double strand DNA breaks. This research has been published (or will be published within the coming year) in high quality, refereed journals; some of it has also been presented at national and international meetings, thereby making it readily available to other workers in the field. Finally, this research has provided scientific training opportunities for five graduate and postdoctoral students and technicians. Of these, two are still working in the lab and three have gone on to other positions in the biomedical sciences. In this manner, our research activities have helped strengthen the country's scientific infrastucture.
