Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5′ to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3′ hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.

[1]  M. Gefter,et al.  DNA Replication , 2019, Advances in Experimental Medicine and Biology.

[2]  T. Steitz,et al.  Structure of the replicating complex of a pol alpha family DNA polymerase. , 2009, Cell.

[3]  E. Kool,et al.  Dynamics of nucleotide incorporation: snapshots revealed by 2-aminopurine fluorescence studies. , 2006, Biochemistry.

[4]  L. Reha-Krantz,et al.  Using 2-aminopurine fluorescence to detect bacteriophage T4 DNA polymerase-DNA complexes that are important for primer extension and proofreading reactions. , 2005, Biochemistry.

[5]  G. Waksman,et al.  Motions of the fingers subdomain of klentaq1 are fast and not rate limiting: implications for the molecular basis of fidelity in DNA polymerases. , 2005, Molecular cell.

[6]  T. Kunkel.  DNA Replication Fidelity* , 2004, Journal of Biological Chemistry.

[7]  C. Kisker,et al.  Lesion (in)tolerance reveals insights into DNA replication fidelity , 2004, The EMBO journal.

[8]  S. Doublié,et al.  Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site , 2004, The EMBO journal.

[9]  R. Rittenhouse,et al.  Characterization of the active site of DNA polymerase β by molecular dynamics and quantum chemical calculation , 2003, Proteins.

[10]  C. M. Joyce,et al.  Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment). , 2003, Biochemistry.

[11]  S. Mandal,et al.  Using 2-Aminopurine Fluorescence to Measure Incorporation of Incorrect Nucleotides by Wild Type and Mutant Bacteriophage T4 DNA Polymerases* , 2002, The Journal of Biological Chemistry.

[12]  M. Tsai,et al.  Use of 2-aminopurine and tryptophan fluorescence as probes in kinetic analyses of DNA polymerase beta. , 2002, Biochemistry.

[13]  W. Konigsberg,et al.  A conserved Tyr residue is required for sugar selectivity in a Pol alpha DNA polymerase. , 2002, Biochemistry.

[14]  Ming-Daw Tsai,et al.  A reexamination of the nucleotide incorporation fidelity of DNA polymerases. , 2002, Biochemistry.

[15]  S. Mandal,et al.  Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. , 2002, Biochemistry.

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

[17]  R. Osman,et al.  Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence. , 2001, Biochemistry.

[18]  K. Hall,et al.  2-Aminopurine fluorescence quenching and lifetimes: role of base stacking. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. O’Donnell,et al.  Sliding clamps: A (tail)ored fit , 2000, Current Biology.

[20]  T. Steitz,et al.  Building a Replisome from Interacting Pieces Sliding Clamp Complexed to a Peptide from DNA Polymerase and a Polymerase Editing Complex , 1999, Cell.

[21]  J. Karam,et al.  Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation. , 1999, Biochemistry.

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

[23]  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.

[24]  Tania A Baker,et al.  Polymerases and the Replisome: Machines within Machines , 1998, Cell.

[25]  T. Steitz,et al.  Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. , 1998, Current opinion in structural biology.

[26]  S. Benkovic,et al.  Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly. , 1998, Biochemistry.

[27]  S. Benkovic,et al.  Dissociation of bacteriophage T4 DNA polymerase and its processivity clamp after completion of Okazaki fragment synthesis. , 1997, Biochemistry.

[28]  X. Zhong,et al.  DNA Polymerase β: Multiple Conformational Changes in the Mechanism of Catalysis† , 1997 .

[29]  C. Jones,et al.  Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins. , 1997, Biochemistry.

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

[31]  S. Benkovic,et al.  The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for holoenzyme complex formation. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  B. Alberts,et al.  Processivity of the Gene 41 DNA Helicase at the Bacteriophage T4 DNA Replication Fork* , 1996, The Journal of Biological Chemistry.

[33]  S. Benkovic,et al.  The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase. , 1995, Biochemistry.

[34]  B. Alberts,et al.  The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment. , 1994, The Journal of biological chemistry.

[35]  S. Benkovic,et al.  Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. , 1992, Biochemistry.

[36]  D. Millar,et al.  Dynamics of mismatched base pairs in DNA. , 1991, Biochemistry.

[37]  L. Nilsson,et al.  Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies. , 1989, Biochemistry.

[38]  C Frieden,et al.  Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system. , 1983, Analytical biochemistry.

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

[40]  S. Benkovic,et al.  Replisome-mediated DNA replication. , 2001, Annual review of biochemistry.

[41]  T. Steitz,et al.  Crystal structure of a pol alpha family replication DNA polymerase from bacteriophage RB69. , 1997, Cell.

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

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

[44]  T. Kunkel,et al.  Fidelity of DNA synthesis. , 1982, Annual review of biochemistry.