Limits...
Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.

Morin JA, Cao FJ, Lázaro JM, Arias-Gonzalez JR, Valpuesta JM, Carrascosa JL, Salas M, Ibarra B - Nucleic Acids Res. (2015)

Bottom Line: To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication.According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state.We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).

View Article: PubMed Central - PubMed

Affiliation: Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, 28049 Madrid, Spain.

Show MeSH

Related in: MedlinePlus

The nucleotide incorporation cycle and alternative kinetic models for DNAP translocation. (A) Minimal kinetic mechanism for processive DNA polymerization. After binding the complementary incoming dNTP to the polymerase–DNA complex (DNAPn), rate limiting changes poise the active site ready for catalysis (Condensation). The polymerase then catalyses the incorporation of the dNTP (Chemistry) and pyrophosphate (PPi) is released. At some point in the polymerization cycle, movement to the next template position occurs. For the following models the steps of dNTP condensation and chemistry were grouped within a single rate limiting step (kcat). (B, C and D) Alternative kinetic models for integrating mechanical translocation within the nucleotide incorporation cycle. Diagrams show the primer-template DNA at the polymerase insertion site (black triangle). Template bases are shown in grey and the incoming nucleotide in green. In each model, the force-dependent translocation occurs at different positions within the cycle: (B) Model 1: translocation (δ) is power-stroked by dNTP binding. (C) Model 2: translocation (δ) is power-stroked by PPi release. (D) Model 3: a Brownian ratchet mechanism where reversible fluctuations between pre- and post- translocated DNAP states (δ) occur after PPi release and before dNTP binding. Rate constants are defined in the main text and Supplementary Data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4402526&req=5

Figure 1: The nucleotide incorporation cycle and alternative kinetic models for DNAP translocation. (A) Minimal kinetic mechanism for processive DNA polymerization. After binding the complementary incoming dNTP to the polymerase–DNA complex (DNAPn), rate limiting changes poise the active site ready for catalysis (Condensation). The polymerase then catalyses the incorporation of the dNTP (Chemistry) and pyrophosphate (PPi) is released. At some point in the polymerization cycle, movement to the next template position occurs. For the following models the steps of dNTP condensation and chemistry were grouped within a single rate limiting step (kcat). (B, C and D) Alternative kinetic models for integrating mechanical translocation within the nucleotide incorporation cycle. Diagrams show the primer-template DNA at the polymerase insertion site (black triangle). Template bases are shown in grey and the incoming nucleotide in green. In each model, the force-dependent translocation occurs at different positions within the cycle: (B) Model 1: translocation (δ) is power-stroked by dNTP binding. (C) Model 2: translocation (δ) is power-stroked by PPi release. (D) Model 3: a Brownian ratchet mechanism where reversible fluctuations between pre- and post- translocated DNAP states (δ) occur after PPi release and before dNTP binding. Rate constants are defined in the main text and Supplementary Data.

Mentions: Replicative DNA polymerases (DNAPs) work as molecular machines that catalyze template-directed DNA replication in a processive manner. These enzymes share a common metal-ion catalytic mechanism for the incorporation of the complementary deoxy-nucleoside triphosphate (nucleotide or dNTP) to the 3′ end of the growing primer strand (1,2). They also present similar structural organization of the polymerization domain, with three subdomains referred to as the thumb, fingers and palm (3). The processive nucleotide incorporation cycle involves a series of conformational changes and intermediate states that couple the chemical steps of the reaction to the mechanical translocation of the polymerase relative to the DNA by one nucleotide at a time (4). In each reaction cycle, the most evident conformational change is the rotation of the fingers subdomain between open and closed conformations. Following the initial binding of the polymerase to the DNA, a minimal model of nucleotide incorporation starts with the binding of an incoming dNTP to form a ternary dNTP–polymerase–DNA complex (Figure 1A). Binding of the complementary dNTP stabilizes the closed conformation of the fingers through a series of intermediate states relevant for nucleotide selection (5–9). Then rate-limiting non-covalent transformations activate the ternary complex to form an active site poised for catalysis (5,10–20). This is immediately followed by a rapid phosphoryl transfer reaction: the new phosphodiester bond is formed with concomitant pyrophosphate (PPi) cleavage from the nucleotide. The cycle is completed by the PPi release and motion of the fingers from the closed to the open state (21,22). During this cycle the polymerase should translocate from the ‘pre-translocated’ state, where the active site is occupied by the newly added nucleotide, to the next 3′-OH primer terminus or ‘post-translocated’ state. The translocation step facilitates the processive movement of the polymerase along the template DNA and it is crucial to maintain genetic integrity. An accurate, highly coordinated stepping is necessary to prevent frame-shift mutations and to modulate the balance toward the ‘editing’ or exonuclease mode in which mismatched bases are excised by the polymerase (16,21,23).


Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.

Morin JA, Cao FJ, Lázaro JM, Arias-Gonzalez JR, Valpuesta JM, Carrascosa JL, Salas M, Ibarra B - Nucleic Acids Res. (2015)

The nucleotide incorporation cycle and alternative kinetic models for DNAP translocation. (A) Minimal kinetic mechanism for processive DNA polymerization. After binding the complementary incoming dNTP to the polymerase–DNA complex (DNAPn), rate limiting changes poise the active site ready for catalysis (Condensation). The polymerase then catalyses the incorporation of the dNTP (Chemistry) and pyrophosphate (PPi) is released. At some point in the polymerization cycle, movement to the next template position occurs. For the following models the steps of dNTP condensation and chemistry were grouped within a single rate limiting step (kcat). (B, C and D) Alternative kinetic models for integrating mechanical translocation within the nucleotide incorporation cycle. Diagrams show the primer-template DNA at the polymerase insertion site (black triangle). Template bases are shown in grey and the incoming nucleotide in green. In each model, the force-dependent translocation occurs at different positions within the cycle: (B) Model 1: translocation (δ) is power-stroked by dNTP binding. (C) Model 2: translocation (δ) is power-stroked by PPi release. (D) Model 3: a Brownian ratchet mechanism where reversible fluctuations between pre- and post- translocated DNAP states (δ) occur after PPi release and before dNTP binding. Rate constants are defined in the main text and Supplementary Data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402526&req=5

Figure 1: The nucleotide incorporation cycle and alternative kinetic models for DNAP translocation. (A) Minimal kinetic mechanism for processive DNA polymerization. After binding the complementary incoming dNTP to the polymerase–DNA complex (DNAPn), rate limiting changes poise the active site ready for catalysis (Condensation). The polymerase then catalyses the incorporation of the dNTP (Chemistry) and pyrophosphate (PPi) is released. At some point in the polymerization cycle, movement to the next template position occurs. For the following models the steps of dNTP condensation and chemistry were grouped within a single rate limiting step (kcat). (B, C and D) Alternative kinetic models for integrating mechanical translocation within the nucleotide incorporation cycle. Diagrams show the primer-template DNA at the polymerase insertion site (black triangle). Template bases are shown in grey and the incoming nucleotide in green. In each model, the force-dependent translocation occurs at different positions within the cycle: (B) Model 1: translocation (δ) is power-stroked by dNTP binding. (C) Model 2: translocation (δ) is power-stroked by PPi release. (D) Model 3: a Brownian ratchet mechanism where reversible fluctuations between pre- and post- translocated DNAP states (δ) occur after PPi release and before dNTP binding. Rate constants are defined in the main text and Supplementary Data.
Mentions: Replicative DNA polymerases (DNAPs) work as molecular machines that catalyze template-directed DNA replication in a processive manner. These enzymes share a common metal-ion catalytic mechanism for the incorporation of the complementary deoxy-nucleoside triphosphate (nucleotide or dNTP) to the 3′ end of the growing primer strand (1,2). They also present similar structural organization of the polymerization domain, with three subdomains referred to as the thumb, fingers and palm (3). The processive nucleotide incorporation cycle involves a series of conformational changes and intermediate states that couple the chemical steps of the reaction to the mechanical translocation of the polymerase relative to the DNA by one nucleotide at a time (4). In each reaction cycle, the most evident conformational change is the rotation of the fingers subdomain between open and closed conformations. Following the initial binding of the polymerase to the DNA, a minimal model of nucleotide incorporation starts with the binding of an incoming dNTP to form a ternary dNTP–polymerase–DNA complex (Figure 1A). Binding of the complementary dNTP stabilizes the closed conformation of the fingers through a series of intermediate states relevant for nucleotide selection (5–9). Then rate-limiting non-covalent transformations activate the ternary complex to form an active site poised for catalysis (5,10–20). This is immediately followed by a rapid phosphoryl transfer reaction: the new phosphodiester bond is formed with concomitant pyrophosphate (PPi) cleavage from the nucleotide. The cycle is completed by the PPi release and motion of the fingers from the closed to the open state (21,22). During this cycle the polymerase should translocate from the ‘pre-translocated’ state, where the active site is occupied by the newly added nucleotide, to the next 3′-OH primer terminus or ‘post-translocated’ state. The translocation step facilitates the processive movement of the polymerase along the template DNA and it is crucial to maintain genetic integrity. An accurate, highly coordinated stepping is necessary to prevent frame-shift mutations and to modulate the balance toward the ‘editing’ or exonuclease mode in which mismatched bases are excised by the polymerase (16,21,23).

Bottom Line: To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication.According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state.We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).

View Article: PubMed Central - PubMed

Affiliation: Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, 28049 Madrid, Spain.

Show MeSH
Related in: MedlinePlus