The Mechanism of Escherichia coli Deoxyribonucleic Acid Polymerase I

Slater, John P.; Tamir, Ilana; Loeb, Lawrence A.; Mildvan, Albert S.

doi:10.1016/s0021-9258(19)44655-3

Cited by 115 publications

(43 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…E. coli DNA polymerase I (Pol I) was purified by the method of Jovin et al (12) as modified by Slater et al (13). DNA polymerase a forms designated C (7.3 S) and D (6.8 S) were purified from calf thymus as described by Holmes et al (14,15).…”

Section: Enzymesmentioning

confidence: 99%

DNA polymerase α and models for proofreading

Abbotts

Loeb

1985

Nucl Acids Res

Self Cite

View full text Add to dashboard Cite

Using a modified system to measure fidelity at an amber site in phi X174, we have employed DNA polymerase alpha to test different mechanisms for proofreading. DNA polymerase alpha does not exhibit the characteristics of "kinetic proofreading" seen with procaryotic polymerases. Polymerase alpha shows no evidence for a "next nucleotide" effect, and added deoxynucleoside monophosphates do not alter fidelity. Pyrophosphate, which increases error rates with a procaryotic polymerase, appears to weakly improve polymerase alpha fidelity. DNA polymerase alpha does exhibit a dramatic increase in error rate in the presence of a deoxycytidine thiotriphosphate (dCTP alpha S), but this enhanced mutagenesis also occurs under conditions where kinetic proofreading should be otherwise defeated. This particular effect with dCTP alpha S appears specific for DNA polymerase alpha and is not seen with the other polymerases tested.

show abstract

Section: Enzymesmentioning

confidence: 99%

DNA polymerase α and models for proofreading

Abbotts

Loeb

1985

Nucl Acids Res

Self Cite

View full text Add to dashboard Cite

show abstract

“…During the initial partial purification of E. coli DNA pol I, it was found that its pol activity absolutely required divalent metal ions and was pH dependent ( Goulian et al, 1968 ; Slater et al, 1972 ; Kornberg and Baker, 1992 ). When the pH deviated from its optimal value or when the concentration of divalent metal ions deviated from their optimal value, the pol activity was reduced.…”

Section: Universal Bell-shaped Pol Activity Curves As a Function Of D...mentioning

confidence: 99%

“…In the most recent studies on a DNA pol from a closely-related bacteriophage, RB69, Konigsberg and others showed that the pol activity was reduced by ∼ 2-fold when [Mn 2+ ] increased from 10 to 20 mM with the estimated slope of −1 in the plot of log activity versus log[Mn 2+ ] ( Vashishtha and Konigsberg, 2016 ; Vashishtha et al, 2016 ). The optimal activity of E. coli DNA pol I is at 0.07 mM [Mn 2+ ], and was reduced by more than 3-fold when [Mn 2+ ] increased to 0.21 mM (3-fold), highlighting the inhibitory effect of Mn 2+ ion on its pol activity ( Figure 4A ) ( Slater et al, 1972 ). The estimated slope of the log activity vs log[Mn 2+ ] plot is −1.0 unit, indicating that there is just a single inhibitory binding site.…”

Section: Universal Bell-shaped Pol Activity Curves As a Function Of D...mentioning

confidence: 99%

“…When the logarithm of the steady-state rate was plotted as a function of [Mg 2+ ], the slope was +2.0 (not 3) before [Mg 2+ ] reached the maximal rate (Bolton et al, 2002). Beyond its optimal concentration, the rate decreased linearly with the increasing concentration, with a slope of -1.0 in both linear and log plots (Slater et al, 1972;Sirover et al, 1979;. The slopes of these plots suggest that pols bind two divalent metal ions for catalytic enhancement and a third divalent metal ion for inhibition according to classic enzymology (Fersht, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…This unique ability for rate enhancement can be attributed to the stabilization provided by this enzyme specifically only to the transition state (TS) relative to the enzyme-substrate (ES) or enzyme-product (EP) complexes as shown by the steady-state kinetics ( Figure 1 ) ( Knowles, 1980 ; Lassila et al, 2011 ). The pol-catalyzed reaction absolutely requires divalent Mg 2+ ions ( Goulian et al, 1968 ; Slater et al, 1972 ; Kornberg and Baker, 1992 ). When the logarithm of the steady-state rate was plotted as a function of [Mg 2+ ], the slope was +2.0 (not 3) before [Mg 2+ ] reached the maximal rate ( Bolton et al, 2002 ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Two-Metal-Ion Catalysis: Inhibition of DNA Polymerase Activity by a Third Divalent Metal Ion

Wang

Konigsberg

2022

Front. Mol. Biosci.

View full text Add to dashboard Cite

Almost all DNA polymerases (pols) exhibit bell-shaped activity curves as a function of both pH and Mg2+ concentration. The pol activity is reduced when the pH deviates from the optimal value. When the pH is too low the concentration of a deprotonated general base (namely, the attacking 3′-hydroxyl of the 3′ terminal residue of the primer strand) is reduced exponentially. When the pH is too high the concentration of a protonated general acid (i.e., the leaving pyrophosphate group) is reduced. Similarly, the pol activity also decreases when the concentration of the divalent metal ions deviates from its optimal value: when it is too low, the binding of the two catalytic divalent metal ions required for the full activity is incomplete, and when it is too high a third divalent metal ion binds to pyrophosphate, keeping it in the replication complex longer and serving as a substrate for pyrophosphorylysis within the complex. Currently, there is a controversy about the role of the third metal ion which we will address in this review.

show abstract

The Role of Metals in Enzyme‐Catalyzed Substitutions at each of the Phosphorus Atoms of ATP

Mildvan

1979

Advances in Enzymology - And Related Areas of Molecular Biology

View full text Add to dashboard Cite

The Mechanism of Escherichia coli Deoxyribonucleic Acid Polymerase I

Cited by 115 publications

References 28 publications

DNA polymerase α and models for proofreading

DNA polymerase α and models for proofreading

Two-Metal-Ion Catalysis: Inhibition of DNA Polymerase Activity by a Third Divalent Metal Ion

The Role of Metals in Enzyme‐Catalyzed Substitutions at each of the Phosphorus Atoms of ATP

Contact Info

Product

Resources

About