Investigations into the Cytotoxic and Mutagenic Effects of DNA Adducts and DNA Structures on DNA Replication
Byproducts of normal cellular processes and environmental toxicants are capable of interacting with cellular DNA, producing a variety of chemical modifications. In addition, when duplex DNA becomes single-stranded, certain DNA sequences are capable of adopting non-B structures potentially causing genomic instability. To avoid activation of cellular checkpoints due to stalled replication forks, cells are equipped with translesion synthesis (TLS) polymerases capable of bypassing DNA lesions arising from these endogenous and exogenous sources. TLS polymerases are able to carry out lesion bypass because they lack the proofreading activity of replicative polymerases and possess larger active sites. In some cases lesion bypass carried out by these polymerases is accurate and efficient, while in other cases, TLS introduce mutations into the genome. Investigations into the cytotoxic and mutagenic effects DNA lesions and non-B structures have on DNA replication are needed to understand their role in cancer, aging and disease.
In these studies, comprehensive experiments were conducted to examine the effect regioisomeric alkylated thymidine lesions have on DNA replication; specifically the role TLS polymerases play in processing these lesions. Produced as a result from exposure to tobacco smoke, alkylated thymidines were shown to be poorly repaired in cells and thus, likely to contribute the mutations detected in lung cancer patients. Using novel LC-MS/MS methods, alkylated thymidines were shown to be both blocking and highly mutagenic to most DNA polymerases in vitro, primarily introducing T&rarrC and T&rarrA mutations. On the other hand, endogenously produced N2-alkyl-2'-deoxyguanosine lesions were shown to be accurately and efficiently bypassed through the combination of two TLS polymerases; pol &kappa or pol &iota inserts the correct dCMP opposite the lesion, and pol &zeta extends past the lesion. In addition, cells deficient in pol &kappa and pol &iota showed elevated levels of G&rarrA and G&rarrT mutations, which were attributed to TLS carried out by pol &eta.
Taking advantage of the competitive replication and adduct bypass (CRAB) assay, we also investigated the effect non-B, G-quadruplex (G4) structures have on DNA replication. The experiments revealed that two G4 sequences located in the promoter regions of the oncogene c-Kit and the proto-oncogene c-Myb were capable of blocking DNA replication. Additionally, E. coli TLS pols II and IV, along with E. coli DNA helicase RecD, were shown to be involved in resolving the c-Kit-G4 sequence.