39115-AC4
Studies of DNA Polymerase Mechanisms by Dynamics Simulations: Conformational Changes in DNA Synthesis and Implications on Fidelity
Annual Report DNA polymerases help maintain the genetic integrity of the cell. Our research involves characterizing the catalytic details of DNA polymerases with medium to low fidelity, namely polymerase beta (pol beta), polymerase lambda (pol lambda), polymerase X (pol X), and polymerase IV (Dpo4), through dynamics simulations, transition path sampling (TPS), quantum mechanics (QM) analysis, and coupled quantum mechanics/molecular mechanics (QM/MM) calculations. Our research also involves elucidating the way DNA and associated proteins fold into compact chromatin fibers using our mesoscopic model of chromatin. Findings: (1) Pol lambda's slippage tendency (Bebenek et al., EMBO Rep., 9:459-464, 2008; Foley and Schlick, J. Am. Chem. Soc., 130:3967-3977, 2008) The origin of pol lambda's unusually high tendency for generating deletion errors is hypothesized to involve DNA slippage, but how this relates to a polymerization cycle is not fully understood. We analyzed crystal structures and MD simulations of Arg517Ala and Arg517Lys pol lambda mutants bound to DNA in the midst of strand slippage. We then compared results from our simulations of various Arg517 mutants to those of wild-type trajectories to elucidate critical interactions that constrain the DNA in the active position. Our results indicated that the mutant residues assume discrete orientations that impact protein-coupled DNA stability by forming unfavorable electrostatic interactions. The extent of DNA movement in our simulations is mutant dependent and mirrors deletion error rates determined for wild-type pol lambda and the Lys and Ala mutants. The DNA motion also reflects the strength of hydrogen bonding interactions between residue 517 and the DNA. Together these results suggest that DNA motion is related to pol lambda's slippage tendency and dNTP-induced DNA repositioning during the normal catalytic cycle is a key to controlling strand slippage during replication. (2) Dpo4's chemical pathways (Wang and Schlick, J. Am. Chem. Soc., 2008, In press DOI:10.1021/ja802215c) Using QM/MM, we investigated possible Dpo4 reaction pathways for the insertion of dCTP opposite 8-oxoguanine, both from a chemistry-competent state and a crystal closed state. It is important to investigate the active-site geometry in the crystal closed state to understand pre-chemistry barriers and interpret the entire enzyme mechanism. The most favorable reaction path was found to involve initial deprotonation of O3'H via two water molecules to O1A of dCTP, overcoming an energy barrier of approximately 20.0 kcal/mol. When the O3'H proton is directly transferred to O1A, the energy barrier is 5 kcal/mol higher. In both reaction pathways, initial deprotonation is the rate-limiting step and a transient trigonal-bipyramidal configuration around PA indicates an associative reaction. Chemical reactions from the less ideal state of the crystal structure have a higher energy barrier (29.0 kcal/mol). The pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the active-site environment. (3) Pol X's incorrect nucleotide insertion (Sampoli Benitez et al., 2008, In submission) To interpret available kinetics data, we analyzed MD simulations of pol X bound to different mismatched nascent base pairs and compared to correct G:C trajectories. Experiments indicate that pol X inserts G:G frequently, A:G moderately, and C:C infrequently. In the G:G simulation, the thumb exhibits a large-scale conformational change that is similar to what occurs in the G:C simulation. In the A:G and G:G (anti) systems, the thumb partially closes, whereas in the C:C system, the thumb remains open. The bases stack instead of pair in the A:G and G:G (anti) systems and become perpendicular to one another in the C:C system. Together all data rationalize pol X's nucleotide insertion tendencies and suggest a geometrical preference of G:G (syn) over G:G (anti) that accounts for pol X's frequent G:G insertion. (4) Chromatin compaction (Arya, Zhang, and Schlick, Biophys. J., 91:133-150, 2006; Arya and Schlick, Proc. Natl. Acad. Sci. USA, 103:16236-16241, 2006; Arya and Schlick, J. Phys. Chem., 126:044107, 2007) We described a new mesoscopic model of oligonucleosomes with flexible histone tails that reproduces experiments better than models with rigid tails. Utilizing a Monte Carlo method, we illuminated the nature of tail/core/DNA interactions at various salt concentrations. Analyses indicate that the H4 histone tails are most important for mediating internucleosomal interactions, followed by H3, H2A, and H2B tails in decreasing order of importance. H3 tails also screen the electrostatic repulsion between the entering/exiting DNA linkers. The H2A and H2B tails are important for mediating fiber/fiber interactions. The postdoctoral fellows, graduate students, and undergraduate students participating in the project receive training in protein/DNA structure and function, modeling and simulation for biomolecules, quantum mechanics, quantum/classical hybrid modeling, high performance computing, applied mathematics, statistical mechanics, reaction kinetics, chemical mechanics, and free energy methods.
