Pre-steady-state kinetics of RB69 DNA polymerase and its exo domain mutants: effect of pH and thiophosphoryl linkages on 3'-5' exonuclease activity.

Abstract:

DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. B family DNA polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than A family DNA polymerases that mainly function in DNA repair. To investigate the basis for these differences, we determined pH-activity profiles for the exonuclease reactions of T4, RB69, and phi29 DNA polymerases as representatives of B family replicative DNA polymerases and the Klenow fragment (KF) as an example of a repair DNA polymerase in the A family. We performed exo assays under single-turnover conditions and found that excision rates exhibited by the B family DNA polymerases were essentially independent of pH between pH 6.5 and 8.5, whereas the exo activity of KF increased 10-fold for each unit increase in pH. Three exo domain mutants of RB69 polymerase had much lower exo activities than the wild-type enzyme and exhibited pH-activity profiles similar to that of KF. On the basis of pH versus activity data and elemental effects obtained using short double-stranded DNA substrates terminating in phosphorothioate linkages, we suggest that the rate of the chemical step is reduced to the point where it becomes limiting with RB69 pol mutants K302A, Y323F, and E116A, in contrast to the wild-type enzyme where chemistry is faster than the rate-determining step that precedes it.

Polymerases:

Topics:

Mutational Analysis, Kinetic Parameters, Structure and Structure/Function, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
RB69 K302A Wang CX2004 kcat 0.0011 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 6.5)
RB69 K302A Wang CX2004 kcat 0.0033 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7)
RB69 K302A Wang CX2004 kcat 0.01 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7.5)
RB69 K302A Wang CX2004 kcat 0.032 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8)
RB69 K302A Wang CX2004 kcat 0.11 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8.5)
RB69 E116A Wang CX2004 kcat 3.9E-05 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 6.5)
RB69 E116A Wang CX2004 kcat 0.00011 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7)
RB69 E116A Wang CX2004 kcat 0.00028 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7.5)
RB69 E116A Wang CX2004 kcat 0.00075 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8)
RB69 E116A Wang CX2004 kcat 0.00053 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8.5)
RB69 Wang CX2004 kcat 17 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrat: 3'-tailed duplex phosphorothioate)
RB69 Wang CX2004 kcat 3 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 6.5)
RB69 Wang CX2004 kcat 5 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7)
RB69 Wang CX2004 kcat 6.6 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7.5)
RB69 Wang CX2004 kcat 7.7 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8)
RB69 Wang CX2004 kcat 8.1 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8.5)
RB69 Wang CX2004 kcat 24 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)
T4 Wang CX2004 kcat 130 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)
T4 Wang CX2004 kcat 91 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 K229A Wang CX2004 kcat 0.18 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)
T4 K229A Wang CX2004 kcat 0.05 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex phosphorothioate substrate)
RB69 Y323F Wang CX2004 kcat 0.14 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)
RB69 Y323F Wang CX2004 kcat 0.00056 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 6.5)
RB69 Y323F Wang CX2004 kcat 0.0018 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7)
RB69 Y323F Wang CX2004 kcat 0.0058 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7.5)
RB69 Y323F Wang CX2004 kcat 0.019 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8)
RB69 Y323F Wang CX2004 kcat 0.056 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8.5)
RB69 Y323F Wang CX2004 kcat 0.0013 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex phosphorothioate substrate)
T4 Y320F Wang CX2004 kcat 0.12 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)
T4 Y320F Wang CX2004 kcat 0.00096 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ( Substrate: 3'-tailed duplex phosphorothioate substrate)
T4 Y320A Wang CX2004 kcat 0.075 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)
T4 Y320A Wang CX2004 kcat 0.0085 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex phosphorothioate substrate)
T4 K299AY320F Wang CX2004 kcat 0.013 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ( Substrate: 3'-tailed duplex phosphorothioate substrate)
T4 K299AY320F Wang CX2004 kcat 0.0005 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
RB69 K302AY323F Wang CX2004 kcat 0.013 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)
RB69 K302AY323F Wang CX2004 kcat 0.00081 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex phosphorothioate substrate)
RB69 K302AY323F Wang CX2004 kcat 0.02 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex phosphorothioate substrate)
RB69 K302AY323F Wang CX2004 kcat 2.1E-05 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 6.5)
RB69 K302AY323F Wang CX2004 kcat 6.5E-05 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7)
RB69 K302AY323F Wang CX2004 kcat 0.0002 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7.5)
RB69 K302AY323F Wang CX2004 kcat 0.00064 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8)
RB69 K302AY323F Wang CX2004 kcat 0.002 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8.5)
Klenow fragment Wang CX2004 kcat 0.02 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ( Substrate: 3'-tailed duplex phosphorothioate substrate)
Klenow fragment Wang CX2004 kcat 0.0017 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 6.5)
Klenow fragment Wang CX2004 kcat 0.0054 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7)
Klenow fragment Wang CX2004 kcat 0.016 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 7.5)
Klenow fragment Wang CX2004 kcat 0.052 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8)
Klenow fragment Wang CX2004 kcat 0.16 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: pH (pH 8.5)
Klenow fragment Wang CX2004 kcat 0.33 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (Substrate: 3'-tailed duplex all-oxygen substrate)

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