Masaaki Moriya - US grants

Genotoxic agents exert their deleterious effects mainly by damaging cellular DNA. In response, cells have evolved several ways to overcome their harmful effects. One of the representative mechanisms is the repair of damaged DNA. Cells attempt to repair DNA damage before the onset of DNA replication or cell division. However, in the undesirable situation, replication of damaged DNA still occurs. Unrepaired DNA lesions often block the progression of DNA synthesis and are the major source of mutations. In this project, mechanisms for cellular responses to unrepaired DNA lesions will be studied using endogenously produced DNA adducts such as 1,N6-ethenodeoxyadenosine and one of the acrolein-derived deoxyguanosine adducts. These adducts are suspected to contribute to aging and cancer. Since they are continuously produced in cellular DNA, it is not unlikely that the cellular replication machinery encounters unrepaired endogenous lesions. If cells did not have any error-free damage tolerance mechanism, the survival and integrity of cellular DNA would depend solely on the efficiency and fidelity of translesion DNA synthesis, and a small number of blocking lesions would be lethal. However, many studies have shown that cells tolerate many unrepaired lesions. On the other hand, if cells had only error-free damage tolerance mechanism, cells would not be mutable by DNA adducts. However, cells are mutable by DNA adducts. Our central hypothesis is that cells respond to unrepaired DNA adducts in an error-free and an error-prone manner. Our preliminary studies have indicated that this is true in E. coli. This organism overcomes synthesis block by error-prone translesion synthesis and error-free daughter strand gap repair. We have demonstrated the existence of these two pathways, at the DNA sequence, using our recently developed approach. This approach utilizes a site-specifically placed single DNA adduct and strand-specific marker sequences to identify the origin of progeny which are derived from various cellular pathways. This new approach will be used to explore error-free and error-prone damage tolerance mechanisms in eukaryotes. The mechanisms will be investigated using plasmid and chromosomal substrates in human cells and yeast. The factors influencing damage tolerance mechanisms and their induction by DNA damage will also be investigated.

DESCRIPTION (provided by applicant): DNA synthesis across unrepaired DNA damage, known as translesion DNA synthesis (TLS), is one of the major mechanisms responsible for chemical mutagenesis; in humans this process is associated with the development of cancer and age-related degenerative diseases. The goal of this research is to elucidate the mechanisms of TLS as they occur in human cells. Several TLS-specialized DNA polymerases (pol), such as pol h, pol k, pol i, pol z and REV1, have been discovered recently; we will determine the role of these enzymes in TLS in human cells. Two exocyclic propanodeoxyguanosine (PdG) DNA adducts and 1,N6-ethenodeoxyadenosine (edA) will be employed as target lesions. We have established the genotoxicity of these adducts in human cells, using a unique shuttle vector system developed in my laboratory. To probe the mechanism for TLS in cells, primer extension studies with purified polymerases will be first conducted to determine their efficiency, fidelity, and coding specificity in vitro. By comparing in vivo and in vitro results, candidate polymerase(s) responsible for TLS events in cells will be identified. The expression of the candidate polymerase gene(s) in human cells will be silenced by RNA interference technology. Such "engineered" cells are then used as hosts for genotoxic analyses. By comparing TLS events in "wild type" and engineered cells, the role and function of candidate polymerase(s) in TLS can be assessed. Extracts of engineered cells will be used to replicate modified plasmid in vitro for biochemical studies. The combination of in vitro TLS studies, in vivo assays for genotoxicity, and RNA interference technology will provide novel insights into the fundamental role and function(s) of TLS polymerases and their contribution to chemical mutagenesis in human cells.

DESCRIPTION (provided by applicant): Cellular DNA is continuously damaged by endogenous and exogenous sources of mutagens. The long-term objectives of this proposal are (i) to understand how unrepaired damage induces mutations that ultimately lead to degenerative diseases, ageing and cancer, and (ii) thereby to contribute to minimize their genotoxic consequences. This knowledge can also be used to maximize the efficacy of cancer chemotherapeutic agents. Recent studies have revealed a new family of mammalian DNA polymerases that are specialized for a DNA synthesis across unrepaired DNA lesions. These low-fidelity polymerases are pol 7, pol :, pol 9, pol 6 and REV1. They play a central role in mutation induction and are thought to be active on different types of DNA lesions. Since they are prone to miscopy undamaged DNA, their activities must be regulated tightly. To study their roles in mutation induction and the mechanism of their regulation, a new experimental approach will be developed, which consists of three major components: DNA containing a chemically defined DNA damage, a plasmid that replicates in mouse cells, and mouse cells, specific genes of which, such as those for specialized DNA polymerases, their regulatory genes and DNA repair genes, are inactivated by gene targeting, thereby the role of the gene of interest is specifically investigated. In addition, experiments, where mutated versions of a gene are introduced into the gene knockout cells to examine their functional complementation, will allow the mechanistic analysis of a translesion synthesis. Typical experiments will be conducted as follows: (i) DNA containing a site-specific DNA lesion is synthesized;(ii) this modified DNA is incorporated into a plasmid;(iii) the modified plasmid is introduced into mouse host cells;(iv) progeny plasmid is recovered and analyzed for the events at the lesion site;and (v) the effect of the gene inactivation on a translesion synthesis is evaluated. With this strategy together with other established techniques such as the in vitro translesion synthesis assay using purified polymerases, the yeast two-hybrid assay for studying protein-protein interaction, and the intracellular localization assay of a polymerase, the mechanism of mammalian mutagenesis will be studied. PUBLIC HEALTH RELEVANCE: Accumulation of DNA damage caused by endogenous, environmental and chemotherapeutic agents is suspected to contribute to ageing, degenerative diseases, and cancer. Hence, it is very important to reveal the cellular mechanism by which unrepaired DNA damage exerts genotoxic effects, such as point mutations and chromosome aberrations, and causes cell death.

? DESCRIPTION (provided by applicant): DNA interstrand crosslinking plays a critical role in the action of certain anti-tumor agents and environmental toxins and also in the pathobiology of endogenous DNA damage as represented by Fanconi anemia (FA). DNA crosslinking cancer chemotherapeutics, including mitomycin C, nitrogen mustards, and platinum compounds, utilize the greater sensitivity of actively growing cancer cells to crosslinking agents. FA, a rare inheritd genetic disease, is caused by mutations in genes regulating replication-associated removal of interstrand DNA crosslinks (ICLs). By linking two DNA strands, ICLs inhibit both DNA replication and transcription. Failure to remove ICL lesions ultimately leads to cell death. Moreover, hereditary breast and ovarian cancers are triggered in individuals with genetic defects in the BRCA genes of FA/BRCA ICL repair pathway. Therefore, it is very critical to reveal the ICL repair mechanism to improve cancer chemotherapy and understand human genetic disease and hereditary cancer proneness. It has long been believed that progression of a replication fork is invariably inhibited when a replication complex encounters an ICL and that fork progression resumes only after the ICL is repaired. However, our recent experiments on ICL repair taking place at a replication fork produced an unexpected result, which is inconsistent with this widely endorsed hypothesis. Our results suggest the formation of a new DNA replication fork downstream of ICL and, additionally, indicate that the recently discovered human Primase-Polymerase, PRIMPOL, plays a critical role in this event. Confirmation of this revolutionary new hypothesis and the demonstration of PRIMPOL's essential role in this process are expected to open important new avenues in our understanding of the mechanism of mammalian ICL repair. To achieve this goal, a single ICL is inserted into plasmid that replicates in one direction in human cells synchronously with the replication of host cells. Progeny plasmids are recovered from cells and analyzed for repair events, which serve as a marker for a new fork formation. The role for PRIMPOL in the formation of a new fork is evaluated by knocking down its activity by the RNA interference technology. For detailed studies, the PRIMPOL gene is destroyed by a newly developed gene knockout technology, and knockout cells are complemented with PRIMPOL mutants. Achievements of these aims will contribute to the improvement of cancer chemotherapeutic drugs and the full understanding of the human genetic diseases associated with a defect in ICL repair.