Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

Towle-Weicksel, Jamie B.; Dalal, Shibani; Sohl, Christal D.; Doublié, Sylvie; Anderson, Karen S.; Sweasy, Joann B.

doi:10.1074/jbc.m114.561878

Cited by 27 publications

(68 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such postcatalytic active site rearrangement and protein conformational change are consistent with those projected through kinetic studies. 36–39 The pyrophosphate dissociation from the crystals observed here was not seen previously after correct nucleotide incorporation onto undamaged DNA by hPol β in crystallo. 12 Furthermore, 8-oxoG in the nicked DNA product after dCTP incorporation was best modeled in both anti - and syn- conformations (Figure S6A), and the incorporated dCMP had poor electron density and stacked, rather than base paired, with 8-oxoG (Figure 1E).…”

Section: Resultssupporting

confidence: 51%

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

et al. 2015

View full text Add to dashboard Cite

One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson–Crick base pair with correct deoxycytidine 5′-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5′-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson–Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg2+ appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3′-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion.

show abstract

Section: Resultssupporting

confidence: 51%

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

et al. 2015

View full text Add to dashboard Cite

show abstract

“…39 To increase specificity and fidelity, pol β utilizes multiple kinetic steps to govern substrate selection. 32,40–42 Previous evidence indicates that, for pol β, chemistry is normally rate-determining for both correct and incorrect nucleotides, though the activation energy barrier for the forward reaction of the incorrect dNTP is much higher. 40,42–46 It has been suggested that the difference in energy results from a distorted active site in the presence of a mismatch.…”

mentioning

confidence: 99%

“…32,40,42 Conformational changes occur upon binding of the correct dNTP, which result in closing of the fingers subdomain and other smaller movements that align the active site for an inline nucleophilic attack with the incoming dNTP, as shown by structural and FRET studies. 13–15,32,33 This then commits the enzyme to chemistry particularly if the reverse fingers opening is a slow reaction compared to the rate of chemistry. 34,35 The chemical reaction requires deprotonation of the 3′-OH to assist with the nucleophilic attack of the 3′-oxygen on Pα of the bound dNTP.…”

mentioning

confidence: 99%

A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase β Cancer-Associated Variant

et al. 2017

Self Cite

View full text Add to dashboard Cite

K289M is a variant of DNA polymerase β (pol β) that has previously been identified in colorectal cancer. The expression of this variant leads to a 16-fold increase in mutation frequency at a specific site in vivo and a reduction in fidelity in vitro in a sequence context-specific manner. Previous work shows that this reduction in fidelity results from a decreased level of discrimination against incorrect nucleotide incorporation at the level of polymerization. To probe the transition state of the K289M mutator variant of pol β, single-turnover kinetic experiments were performed using β,γ-CXY dGTP analogues with a wide range of leaving group monoacid dissociation constants (pKa4), including a corresponding set of novel β,γ-CXY dCTP analogues. Surprisingly, we found that the values of the log of the catalytic rate constant (kpol) for correct insertion by K289M, in contrast to those of wild-type pol β, do not decrease with increased leaving group pKa4 for analogues with pKa4 values of <11. This suggests that one of the relative rate constants differs for the K289M reaction in comparison to that of the wild type (WT). However, a plot of log(kpol) values for incorrect insertion by K289M versus pKa4 reveals a linear correlation with a negative slope, in this respect resembling kpol values for misincorporation by the WT enzyme. We also show that some of these analogues improve the fidelity of K289M. Taken together, our data show that Lys289 critically influences the catalytic pathway of pol β.

show abstract

“…Crystal structures with open (Figure 5d), closed (Figure 5e), and intermediate conformations have been obtained with mismatched or incorrect dNTPs, such as dA that cannot form a correct base pair with dG of the DNA substrate. Previous fluorescence studies [118,121,122] showed movements in Pol β upon incorporation of dNTPs and cofactors, while more recent studies [108] indicate the rapid movement (ms timescale) of the fingers domain upon binding to correct dNTPs. Incorporation of incorrect dNTPs did not induce these conformational changes.…”

Section: Case Studiesmentioning

confidence: 99%

“…The chemical step of catalysis is followed by the release of pyrophosphate (PP i ) and the DNA product (DNA n +1 ) (step 4). Figure adapted from [108,109]. …”

Section: Figurementioning

confidence: 99%

Role of Conformational Motions in Enzyme Function: Selected Methodologies and Case Studies

2016

View full text Add to dashboard Cite

It is now common knowledge that enzymes are mobile entities relying on complex atomic-scale dynamics and coordinated conformational events for proper ligand recognition and catalysis. However, the exact role of protein dynamics in enzyme function remains either poorly understood or difficult to interpret. This mini-review intends to reconcile biophysical observations and biological significance by first describing a number of common experimental and computational methodologies employed to characterize atomic-scale residue motions on various timescales in enzymes, and second by illustrating how the knowledge of these motions can be used to describe the functional behavior of enzymes and even act upon it. Two biologically relevant examples will be highlighted, namely the HIV-1 protease and DNA polymerase β enzyme systems.

show abstract

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

Cited by 27 publications

References 60 publications

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase β Cancer-Associated Variant

Role of Conformational Motions in Enzyme Function: Selected Methodologies and Case Studies

Contact Info

Product

Resources

About