DNA Polymerase Fidelity: A Computational Study of Pol lambda and Other X-Family Enzymes

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 lambda's much higher single-base deletion error rate and use of large DNA shifting in contrast to pol beta's 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 lambda's 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 beta's 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 lambda's 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.