Biochemical Mechanisms of Polymerase Enzyme Function in DNA Damage Tolerance

Abstract

Cancer is one of the main causes of death worldwide. It arises from single cells that transform from into tumor cells by accumulating mutations formed upon exposure to chemicals. Each human cell undergoes several thousand DNA-damaging events every day, resulting in the formation of DNA adducts, a key initiating step in the genotoxic mechanism of carcinogenesis. DNA alkylation occurs frequently and cells have evolved protective DNA repair mechanism that counteract DNA damage. However, not all DNA adducts are recognized by the cellular DNA repair pathways and some might evade the repair-process. If DNA adducts persist to the S-phase of the cell cycle, they have the potential to disrupt and stall the high fidelity DNA replication machinery, resulting in replication fork collapse, double-strand breaks, and genomic instability. DNA damage tolerance can rescue DNA replication by allowing replication past DNA adducts in a process called translesion DNA synthesis (TLS). Human cells contain specialized TLS polymerases capable of accommodating DNA adducts and replicating past sites of DNA damage, but typically have low fidelity and processivity and may be accountable for mutations. An important class of DNA damaging chemicals are alkylating agents that can generate the highly mutagenic O6-methylguanine (O6-MeG) or O6-carboxymethlyguanine (O6-CMG). Both adducts are of high interest due to their potential connection with colon carcinogenesis associated with diets high in processed or red meat. Therefore, understanding how human polymerases process particular DNA adducts is an important aspect of elucidating carcinogenesis mechanisms of genotoxins. Chapter 1 contains the scientific background of this Ph.D. thesis including an introduction to the structure and function of DNA and its susceptibility to chemical alterations. The chapter also covers aspects of the biochemical processing of DNA adducts by DNA repair and tolerance pathways and their contribution to mutagenesis. Finally, the structure and function of DNA polymerases are introduced and mechanisms governing the fidelity of polymerases are discussed. In Chapter 2, the mutagenic potential of the potential colon cancer-related DNA adduct O6-CMG was investigated. The enzymatic processing of O6-CMG was characterized by human TLS Polymerases η, ι, κ, ζ and the replicative Pol δ and their bypass-proficiencies were compared on the basis of steady-state kinetics experiments. The results revealed characteristic dNTP incorporation patterns for the polymerases tested and provided first in vitro evidence that O6-CMG is a substrate for TLS but might contribute to GC to AT and GC to TA mutations. These findings suggest a chemical basis for mutational signatures frequently observed in colon cancer In Chapter 3 a structure-activity relationship study was carried out to identify structural properties of O6-CMG that affect the fidelity of DNA polymerases. A post-synthetic DNA modification strategy was used to generate O6-CMG analogs with varied size and electrostatic properties. The influence of structural variations of the CMG analogs on polymerase fidelity was assessed with primer extension experiments. The results demonstrated that Pol κ was sensitive to structural variations and more mutagenic in the bypass of O6-CMG analogs with increased steric bulk or a positive charge compared to the bypass of O6-CMG. In contrast, Pol ι, and in particular Pol η, were marginally affected by steric effects and showed little sensitivity to charge related changes of the analogs. In Chapter 4, the effect of interbase- and DNA-Pol hydrogen bonding interactions on the fidelity and processivity of DNA polymerase ζ were investigated. Canonical and synthetic bases with diverse hydrogen bond donor and acceptor capabilities were placed opposite both guanine and a series of methylated guanine adducts to systematically test the influence of differential hydrogen bonding on the activity of polymerase ζ. The results demonstrated that terminal base pairs with the highest proclivity for H-bonding were most efficiently extended whereas when no H-bonding was possible, extension was hampered. In addition, the primer extension studies combined with computational modeling suggested that minor groove hydrogen bonding between a conserved lysine residue in the polymerase and a carbonyl group in the primer strand are important for successful primer extension. These data provided new insight regarding contributions of hydrogen bonding interactions to the correct extension from alkylated DNA adducts by TLS polymerases ζ. Chapter 5 summarizes the most important results of the doctoral work and presents an outlook on future studies. The findings of this thesis suggest a candidate chemical basis for mutations observed in colon cancer and offer insights in chemical interactions important for Pol fidelity in the bypass of alkylated guanine adducts. The Appendix contains a manuscript addressing a complementary aspect of nucleic acids chemistry and biochemistry concerning the influence of torsional constraints of Cas9 substrates. This work was carried out during a research visit to the University of Kyoto, Japan, and was realized by using DNA self-assembly structures. Cas9 binding to different DNA structures was characterized with single-molecule resolution. Additionally, the ability of Cas9 to cleave the torsional constrained or unconstrained dsDNA was compared based on qPCR experiments and the reaction was followed by high-speed atomic force microscopy. The results revealed that highly ordered and constrained DNA structures could be obstacles for Cas9.