The mechanism of the translocation step in DNA replication by DNA polymerase I: a computer simulation analysis.

High-fidelity DNA polymerases copy DNA rapidly and accurately by adding correct deoxynucleotide triphosphates to a growing primer strand of DNA. Following nucleotide incorporation, a series of conformational changes translocate the DNA substrate by one base pair step, readying the polymerase for the next round of incorporation. Molecular dynamics simulations indicate that the translocation consists globally of a polymerase fingers-opening transition, followed by the DNA displacement and the insertion of the template base into the preinsertion site. They also show that the pyrophosphate release facilitates the opening transition and that the universally conserved Y714 plays a key role in coupling polymerase opening to DNA translocation. The transition involves several metastable intermediates in one of which the O helix is bent in the vicinity of G711. Completion of the translocation appears to require a gating motion of the O1 helix, perhaps facilitated by the presence of G715. These roles are consistent with the high level of conservation of Y714 and the two glycine residues at these positions. It is likely that a corresponding mechanism is applicable to other polymerases.

[1]  Smita S. Patel,et al.  Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. , 1991, Biochemistry.

[2]  Samuel H. Wilson,et al.  Polymerase beta simulations suggest that Arg258 rotation is a slow step rather than large subdomain motions per se. , 2002, Journal of molecular biology.

[3]  P Argos,et al.  An attempt to unify the structure of polymerases. , 1990, Protein engineering.

[4]  T. Steitz,et al.  Crystal Structure of a pol α Family Replication DNA Polymerase from Bacteriophage RB69 , 1997, Cell.

[5]  M. O’Donnell,et al.  Fidelity of Escherichia coli DNA Polymerase III Holoenzyme , 1997, The Journal of Biological Chemistry.

[6]  T. Carell,et al.  Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase , 2004, Nature.

[7]  W. Greenleaf,et al.  Direct observation of base-pair stepping by RNA polymerase , 2005, Nature.

[8]  Gabriel Waksman,et al.  Structure and mechanism of DNA polymerases. , 2005, Advances in protein chemistry.

[9]  Thomas A Steitz,et al.  The Structural Mechanism of Translocation and Helicase Activity in T7 RNA Polymerase , 2004, Cell.

[10]  Sean J. Johnson,et al.  Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[12]  M. Karplus,et al.  Dependence of DNA polymerase replication rate on external forces: a model based on molecular dynamics simulations. , 2004, Biophysical journal.

[13]  Laxmikant V. Kale,et al.  NAMD2: Greater Scalability for Parallel Molecular Dynamics , 1999 .

[14]  T. Schlick,et al.  Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition path sampling of DNA polymerase beta's closing. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[16]  T. Steitz,et al.  Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP , 2020, Nature.

[17]  B. Brooks,et al.  Constant pressure molecular dynamics simulation: The Langevin piston method , 1995 .

[18]  Alexander D. MacKerell,et al.  All‐atom empirical force field for nucleic acids: I. Parameter optimization based on small molecule and condensed phase macromolecular target data , 2000 .

[19]  P. Cramer,et al.  Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution , 2001, Science.

[20]  K. Severinov,et al.  Crystal Structure of Thermus aquaticus Core RNA Polymerase at 3.3 Å Resolution , 1999, Cell.

[21]  David Chandler,et al.  Transition path sampling: throwing ropes over rough mountain passes, in the dark. , 2002, Annual review of physical chemistry.

[22]  S. Doublié,et al.  Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution , 1998, Nature.

[23]  Thomas A. Steitz,et al.  Structure of the Replicating Complex of a Pol α Family DNA Polymerase , 2001, Cell.

[24]  S. Benkovic,et al.  DNA polymerase fidelity: kinetics, structure, and checkpoints , 2004 .

[25]  T. Kunkel,et al.  Side Chains That Influence Fidelity at the Polymerase Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)* , 1999, The Journal of Biological Chemistry.

[26]  Alexander D. MacKerell,et al.  All‐atom empirical force field for nucleic acids: II. Application to molecular dynamics simulations of DNA and RNA in solution , 2000 .

[27]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[28]  Sean J. Johnson,et al.  Structures of Mismatch Replication Errors Observed in a DNA Polymerase , 2004, Cell.

[29]  Jürgen Schlitter,et al.  Targeted Molecular Dynamics Simulation of Conformational Change-Application to the T ↔ R Transition in Insulin , 1993 .

[30]  L. Beese,et al.  Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution. , 1997, Structure.

[31]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[32]  T. Steitz.  DNA Polymerases: Structural Diversity and Common Mechanisms* , 1999, The Journal of Biological Chemistry.

[33]  Arup K Chakraborty,et al.  Atomistic understanding of kinetic pathways for single base-pair binding and unbinding in DNA , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  L. Beese,et al.  The structural basis for the mutagenicity of O6-methyl-guanine lesions , 2006, Proceedings of the National Academy of Sciences.

[35]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[36]  K. Johnson,et al.  Conformational coupling in DNA polymerase fidelity. , 1993, Annual review of biochemistry.

[37]  Gabriel Waksman,et al.  Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation , 1998, The EMBO journal.

[38]  James R. Kiefer,et al.  Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal , 1998, Nature.

[39]  Martin Karplus,et al.  Simulation of conformational transitions by the restricted perturbation-targeted molecular dynamics method. , 2005, The Journal of chemical physics.

[40]  T. Steitz,et al.  Function and structure relationships in DNA polymerases. , 1994, Annual review of biochemistry.

[41]  Alexander D. MacKerell,et al.  A molecular mechanics force field for NAD+ NADH, and the pyrophosphate groups of nucleotides , 1997, J. Comput. Chem..

[42]  A. Warshel,et al.  Computer simulation studies of the fidelity of DNA polymerases , 2003, Biopolymers.

[43]  S. Benkovic,et al.  A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity. , 1991, Biochemistry.