DNA repairs and replication depend on the activity of specialized enzymes called DNA polymerases. These vary in size and complexity, but all possess a core grouping of subdomains that, like a hand, can grasp DNA and catalyze the insertion of nucleotide building blocks. Fidelity is the ability of a DNA polymerase to correctly match the original templating sequence. Any error introduced into the DNA sequence can be very deleterious and give rise to diseases such as cancer. This research centers on understanding the catalytic cycle of X-family DNA polymerases involved in oxidative DNA damage and double-strand break repairs. Specifically, it focuses on DNA polymerase lambda and its relationship to DNA polymerase beta. It investigates pol lambdas much higher single-base deletion error rate and use of large DNA shifting in contrast to pol betas large-scale thumb motions. We discover similarities in the motions of gate-keeping residues utilized by these enzymes and between the role played by Arg517 in pol lambdas fidelity and Arg283 in pol beta. Our mutation studies of Arg517 show how this residue stabilizes DNA through favorable electrostatic interactions. We relate the extent of DNA shifting in the mutants to deletion error rates, which supports a role for DNA slippage mediated deletion errors. Pol betas comparative lack of DNA motion would help to prevent deletions. Interestingly, studies of pol lambda mis-match complexes exhibit different Arg517/DNA interactions and more DNA shifting； we propose that these motions affect pol lambdas ability to insert incorrect nucleotides. Our studies of pol lambda/misaligned complexes reveal a network of positively-charged thumb residues that create strong interactions with misaligned DNA. Our energetic analyses reinforce this picture by showing that pol lambda/misaligned complexes are more stable than aligned DNA complexes. Our modeling of pol beta/misaligned DNA reveals no comparable network of thumb/DNA interactions, which agrees with its lower deletion error rate. Based on our research and other studies, we relate polymerase function and fidelity. This analysis supports the importance of intrinsic DNA polymerase motions and identifies key features of the polymerase/nucleotide interactions. We suggest a hybrid conformational selection/induced-fit model to better describe nucleotide binding. Finally, we show how this understanding may improve small molecule screening and targeted disease therapies.

Oxidatively generated damage to DNA induced by photosensitization of the pyrene-like aromatic residue Py) derived from the covalent binding of 7,8-dihydrodiol-9,10-oxide-benzo[a]pyrene BPDE) to N2-guanine in the DNA sequence context 5-dCAT[G 1Py]CG2TCCTAC) M) in aerated solutions was monitored from the initial hole injection step to the formation of chemical end-products using HPLC, mass spectrometry, and high-resolution gel electrophoresis. Hole injection into the DNA was initiated by two-photon excitation of the Py residue with intense 355 nm laser pulses, thus producing the Py&middot；＋ radical cation and hydrated electrons e h). The dissolved molecular oxygen captures the hydrated electrons yielding the superoxide radical anion. The oxidative damage occurs mostly at the modified guanine residue G1Py and, to a lesser extent, to the more distant guanine G2. The major products are the 2-amino-5-[2-deoxy-beta-D- erythro-pentofuranosyl) amino]-4H-imidazol-4-one dIz) and M＋16 lesion which has a mass larger by 16 Da than the mass of G 1Py. The structure of the M＋16 product includes an unusual structure with a ruptured non-aromatic cyclohexenyl ring reported here for the first time. The formation of this product limits the efficiency of hole injection into the duplex and therefore the damage observed at G2. The formation of tandem lesions is observed even at low levels of irradiation corresponding to “single-hit” conditions when less than &sim；10% of the oligonucleotide strands are damaged. It is shown that oxidative damage at G1 must occur prior to that of G2 tandem lesion). The formation of products in the unmodified complementary strand Mc in M&middot；Mc duplexes is &sim；10 times smaller than in the modified strand M. In the second part of this dissertation I successfully synthesized the 2-deoxynucleoside and oligonucleotide adducts of benzo[a]pyrene-7,8-dione BPQ) which is a potentially carcinogenic metabolite of benzo[a]pyrene different from the well known BPDE. Two novel BPQ-dC1,2 adducts were discovered in addition to the previously known four BPQ-dG, two BPQ-dA, and two other BPQ-dC adducts. The thermal stabilities of double stranded BPQ-modified DNA were substantially lower than those of the unmodified duplexes. The sites of BPQ-modification in oligonucleotide were determined by exonuclease sequencing combined with MALDI-TOF MS method.

Molecular dynamics (MD) simulations offer researchers a high resolution window into the atomic world. Though far from perfect, they provide researchers with a direct method for probing molecular scale processes. We have used MD to address a range of questions which span three fundamentally different topics. First, we used MD to elucidate the native state energy landscape of a small globular protein. We are able to identify a subset of direct and water-mediated contacts which may be responsible for sculpting this landscape. Next, we turn to the topic of chromatin, specifically, nucleosomal electrostatics. Using a combination of all-atom simulations and Poisson-Boltzmann calculations we are able to observe and explain counterion condensation levels and distribution patterns around the nucleosome core particle. Additionally, our results reveal the significant solvent accessibility of the core particle. Finally, we use all-atom simulations to examine small, artificial, coiled-coil peptides under development for use in light harvesting antennae. Photosensitive chromophores can be tethered to these peptides at a range of sites, and we observe that the choice of these sites plays an important role in regulating energy transfer. In addition, the flexibility of the tethers imbues the chromophores with a large amount of conformational freedom, making their dynamics important for regulating energy transfer.