UC Santa Cruz

DNA encodes the genetic material that makes life possible but it is semi labile and prone to damage. In part, what makes DNA such a reliable genetic storage material is that organisms have developed extensive methods in order to repair damaged DNA and to preserve its integrity. Paradoxically though, genetic instability is essential for survival and one of the major causes of genetic instability is DNA damage sustained through endogenous and exogenous means. Without genetic instability, speciation, evolution, adaptation, and some aspects of aging would not occur. Unfortunately genetic instability does have negative aspects as well such as genetic diseases, cancer, and death. Therefore the interplay between DNA damage and repair is truly a double-edged sword.

E. coli is a valuable model organism for studying DNA damage, repair and replication due to its fast generation time and genetic malleability. Chapters 2-4 of my dissertation widely differ in their scope and aims but all the chapters take advantage of different E. coli genetic and complementation systems in order to answer various questions about DNA damage, DNA repair proteins, or DNA replication.

One particular form of DNA damage used extensively by chemotherapeutic agents is known as reactive DNA methylation. Reactive DNA methylation is defined, in its simplest form, as the non-enzymatic addition of methyl groups (-CH3) to various positions on DNA nucleotides (2). Reactive DNA methylation lesions can either be innocuous, cytotoxic, or mutagenic depending on exactly where on the nucleobase the lesion occurs. O⁶-MeG (methyl-guanine) lesions are particularly cytotoxic to humans and the only protein in the entire human body that directly repairs these lesions is called O⁶ Methyl Guanine Methyl Transferase (MGMT) (3). In Chapter 2 of my thesis I take advantage of an E. coli genetic system that is deficient in its ability to repair O⁶-MeG lesions and determine the repair abilities of all ten non-synonymous MGMT mutants detected in the human population. The results display the O⁶-MeG repair profile of all ten non-synonymous MGMT mutants as well as give new insight into the structure of the MGMT protein.

During the first step of a DNA repair process known as Base Excision Repair (BER) DNA repair enzymes known as glycosylases recognize and remove damaged nucleotides. Base excision repair is not a perfect repair process because at high concentrations glycosylases have been known to remove undamaged bases and, in addition, the resulting abasic site can become problematic if not processed quickly by other enzymes (4). Glycosylases can be divided into two categories: those that are constitutively expressed and those that are inducible. In Chapter 3 of my thesis I utilize an E. coli strain that is devoid of reactive methylation specific glycosylases to understand why organisms have evolved both constitutively expressed and inducible glycosylases. The results show that the activity levels towards substrates in constitutively expressed glycosylases are limited so as not to have a negative effect on the fitness of the organism.

DNA polymerase I (Pol I) is one of two replicative polymerases in E. coli. ColE1 plasmids are used extensively in molecular biology. During ColE1 plasmid replication Pol I initiates leading strand synthesis and also processes Okazaki fragments, Pol III replicates the rest of the plasmid. In Chapter 4 of my thesis I utilize an E. coli strain with a temperature sensitive endogenous Pol I complemented with an error-prone Pol I in order to determine, with high resolution, where on ColE1 plasmids Pol I is replicating (1). The results show exactly where the switch between Pol I and Pol III occurs as well as the sites of Okazaki processing on the lagging strand. In addition the results show that a small fraction of plasmids are replicated exclusively by Pol I and that Pol I plays a far greater role in replication termination than previously thought. These latter results support earlier reports showing a functional redundancy between Pol I and Pol III and bring up interesting questions regarding the role of Pol I during chromosomal replication.