Advances in Structural and Single-Molecule Methods for Investigating DNA Lesion Bypass and Repair Polymerases

Raper, Austin T.; Reed, Andrew J.; Gadkari, Varun V.; Suo, Zucai

doi:10.1021/acs.chemrestox.6b00342

Cited by 3 publications

(9 citation statements)

References 85 publications

(240 reference statements)

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“…The polymerase complexes relevant to panel (A) are depicted and the corresponding enzyme forms relevant to those shown in Scheme A are shown in parentheses. Reproduced from ref . Copyright 2017 American Chemical Society.…”

Section: Kinetic and Structural Mechanism Of Dna Polymerasesmentioning

confidence: 99%

“…Other unique properties of DNA polymerases made implementation of timeresolved X-ray crystallography successful including the relative ease of crystallizing pre-catalytic complexes, the ability to achieve high resolution diffraction data, and the limited impact of conformational heterogeneity or dynamics on crystal integrity during reaction progression. 225 While still a relatively new method (e.g., the first reports for DNA polymerases appeared only approximately five years ago), researchers have already enjoyed success in utilizing the time-resolved structural technique to uncover new details of structure and function relationships of DNA polymerases. In the coming years, we expect that more details of the DNA polymerase mechanism, extending beyond just the X-and Y-families, will be uncovered as more investigators adapt this powerful methodology.…”

Section: Post-chemistry Steps Of Nucleotide Incorporationmentioning

confidence: 99%

“…The ability to follow an enzymatic reaction at atomic resolution has been sought after for many years by biochemists and structural biologists. 225 Static crystal structures of complexes carefully designed to mimic reactant-, intermediate-, and product-states can, at best, only offer hints of the actual reaction mechanism. With the advent of time-resolved X-ray crystallography (Figure 5), much of the ambiguity that accompanies the interpretation of static crystal structures is replaced with clear insight into the chemical mechanism of a particular enzymecatalyzed reaction.…”

Section: Post-chemistry Steps Of Nucleotide Incorporationmentioning

confidence: 99%

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Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

2018

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Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyltransfer (i.e., chemistry), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallographic studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidence for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.

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Section: Kinetic and Structural Mechanism Of Dna Polymerasesmentioning

confidence: 99%

Section: Post-chemistry Steps Of Nucleotide Incorporationmentioning

confidence: 99%

Section: Post-chemistry Steps Of Nucleotide Incorporationmentioning

confidence: 99%

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Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

2018

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“…In addition to these in vitro techniques, super-resolution imaging has provided unprecedented insight into the mechanisms of DNA replication in the physiological environment of the cell. − Below, we briefly discuss these in vitro single-molecule techniques. For additional details, we direct the reader to more in-depth reviews on this topic − and a recent review discussing lesion bypass mechanisms by X-ray crystallography and smFRET …”

Section: Introductionmentioning

confidence: 99%

“…For additional details, we direct the reader to more in-depth reviews on this topic 54−64 and a recent review discussing lesion bypass mechanisms by X-ray crystallography and smFRET. 65 Two force-based methods that have been used to investigate DNA Pol mechanisms at a damaged site are optical and magnetic tweezers. In typical optical tweezers trapping experiments, a DNA molecule is tethered to polystyrene beads trapped by a highly focused laser beam (Figure 2A).…”

Section: Introductionmentioning

confidence: 99%

Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution

Gahlon

Romano

Rueda

2017

Chem. Res. Toxicol.

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Faithful replication of DNA is a critical aspect in maintaining genome integrity. DNA polymerases are responsible for replicating DNA, and high-fidelity polymerases do this rapidly and at low error rates. Upon exposure to exogenous or endogenous substances, DNA can become damaged and this can alter the speed and fidelity of a DNA polymerase. In this instance, DNA polymerases are confronted with an obstacle that can result in genomic instability during replication, for example, by nucleotide misinsertion or replication fork collapse. It is important to know how DNA polymerases respond to damaged DNA substrates to understand the mechanism of mutagenesis and chemical carcinogenesis. Single-molecule techniques have helped to improve our current understanding of DNA polymerase-mediated DNA replication, as they enable the dissection of mechanistic details that can otherwise be lost in ensemble-averaged experiments. These techniques have also been used to gain a deeper understanding of how single DNA polymerases behave at the site of the damage in a DNA substrate. In this review, we evaluate single-molecule studies that have examined the interaction between DNA polymerases and damaged sites on a DNA template.■ CONTENTS

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Brief Research on the Biophysical Study and Anticancer Behavior of Pt(II) Complexes: Their DNA/BSA Binding, Molecular Docking, and Cytotoxic Property

et al. 2022

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The potent bidentate carrier ligand 2-picolylamine (pic) has been used to synthesize Pt(II) complexes to know their bioactivity and anticancer property as reflected by PASS prediction software. The dichloro Pt(II) complex [Pt(pic)Cl 2 ], Pt-1, and its hydrolyzed diaqua complex [Pt(pic)(OH 2 ) 2 ] 2+ , Pt-2, were synthesized. The thiol-containing Pt(II) complexes [Pt(pic)(Lcys)] + , Pt-3, and [Pt(pic)(L-ac-L-cy)] + , Pt-4, were synthesized from Pt-2, which was obtained from hydrolysis of Pt-1. Their biomolecular interactions with BSA and DNA were executed by spectroscopic methods, and their cytototoxic property was tested by the MTT assay. In vitro biomolecular interactions of Pt(II) complexes with BSA and DNA were investigated by different spectroscopic and viscosity measurement methods for their pharmacokinetic and pharmacodynamic importance. The conformational change of BSA in the presence of a drug candidate was studied by Forster resonance energy transfer calculation and synchronous and three-dimensional fluorescence spectroscopic studies. A theoretical approach on optimization structures, highest occupied molecular orbital−lowest unoccupied molecular orbital energy, global reactivity parameters, time-dependent density functional theory, and molecular docking with BSA and DNA was executed to strengthen and support the experimental observations. In vitro cytotoxic profiles of the complexes like the anticancer activity and their level of reactive oxygen species production were brought under consideration on A549 cancer cells and the normal human embryonic kidney cell line HEK-293. The cytotoxic property was compared with that of the recognized anticancer drug cisplatin.

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Advances in Structural and Single-Molecule Methods for Investigating DNA Lesion Bypass and Repair Polymerases

Cited by 3 publications

References 85 publications

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution

Brief Research on the Biophysical Study and Anticancer Behavior of Pt(II) Complexes: Their DNA/BSA Binding, Molecular Docking, and Cytotoxic Property

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