Base Pair Opening in a Deoxynucleotide Duplex Containing a <i>cis-syn</i> Thymine Cyclobutane Dimer Lesion

Wenke, Belinda B.; Huiting, Leah N.; Frankel, Elisa B.; Lane, Benjamin F.; Núñez, Megan E.

doi:10.1021/bi401312r

Cited by 7 publications

(13 citation statements)

References 59 publications

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“…In DNA1 and DNA3, the cs CPD is flanked by GC base pairs on either side, whereas it is flanked by one GC (3′ of cs CPD) and one AT (5′ of cs CPD) base pair in DNA2. Work by the Stanley group on melting temperatures of cs CPD-dsDNA with fluorescent bases has shown that the base pairs on the 5′-side of the CPD, including the 5′-T of the cs CPD, are slightly less stable than those on the 3′-side of the CPD. ,, These results are corroborated by a recent NMR study that shows that the 5′-TA base pair of the CPD is more labile than its 3′-TA base pair . Therefore, it seems that the flanking 5′-TA base pair of the cs CPD in DNA2 destabilizes the cs CPD-dsDNA slightly more than a flanking GC base pair.…”

Section: Discussionmentioning

confidence: 81%

“…The role of the enzyme in base flipping is not completely clear. A computational study of cs CPD-containing DNA and a recent isothermal calorimetry study of photolyase–substrate binding provided support for binding of photolyase to an extrahelical CPD that spontaneously flips out of the double helix. , However, a recent NMR study of base flipping in cs CPD-containing DNA argued against the spontaneous movement of the cs CPD into an extrahelical position as a viable mechanism …”

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confidence: 99%

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Enzyme–Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer

2015

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Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) that are formed on DNA upon exposure of cells to ultraviolet light. The light-driven electron-transfer mechanism by which photolyase catalyzes the CPD monomerization after the enzyme-substrate complex has formed has been studied extensively. However, much less is understood about how photolyase recognizes CPDs on DNA. It has been clearly established that photolyase, like many other DNA-repair proteins, requires flipping of the CPD site into an extrahelical position. Photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base. In this paper, we perform direct measurements of photolyase binding to CPD-containing undecamer DNA that has been labeled with a fluorophore. We find that the association constant of ∼2 × 10(6) M(-1) is independent of the location of the CPD on the undecamer DNA. The binding kinetics of photolyase are best described by two rate constants. The slower rate constant is ∼10(4) M(-1) s(-1) and is most likely due to steric interference of the fluorophore during the binding process. The faster rate constant is on the order of 2.5 × 10(5) M(-1) s(-1) and reflects the binding of photolyase to the CPD on the DNA. This result indicates that photolyase finds and binds to a CPD lesion 100-4000 times slower than other DNA-repair proteins. In light of the existing literature, we propose a mechanism in which photolyase recognizes a CPD that is flipped into an extrahelical position via a three-dimensional search.

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Section: Discussionmentioning

confidence: 81%

mentioning

confidence: 99%

Enzyme–Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer

2015

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“…A study of CPDs in T-tracts in a rotationally phased nuclesosome core particle found, however, that repair by photolyase is greatly inhibited relative to free DNA and that sites of more efficient repair do not correlate with their rotational position ( 40 ) indicating that steric and other factors may be more important in CPD recognition by photolyase. Another possibility is that a base opposite the CPD is flipping out as observed for T4 endonuclease V binding to a CPD ( 41 ), or as deduced from a recent proton exchange nuclear magnetic resonance study on base pair opening in a CPD-containing DNA duplex ( 42 ). This possibility is less likely, however, since the bases opposite a CPD in position 6 which has the fastest deamination rate, have the lowest temperature factors and would be predicted to have the least flexibility since they are held against the histone core surface.…”

Section: Discussionmentioning

confidence: 99%

Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle

Song

Cannistraro

Taylor

2014

Nucleic Acids Research

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Sunlight-induced C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C or 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by DNA polymerase η and defines a probable mechanism for the origin of UV-induced C to T mutations. We have now determined the photoproduct formation and deamination rates for 10 consecutive T=mCG CPDs over a full helical turn at the dyad axis of a nucleosome and find that whereas photoproduct formation and deamination is greatly inhibited for the CPDs closest to the histone surface, it is greatly enhanced for the outermost CPDs. Replacing the G in a T=mCG CPD with A greatly decreased the deamination rate. These results show that rotational position and flanking sequence in a nucleosome can significantly and synergistically modulate CPD formation and deamination that contribute to C to T mutations associated with skin cancer induction and may have influenced the evolution of the human genome.

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“…where: α is known to be approximately equal to unity for the ammonia base catalyst (Wenke, Huiting, Frankel, Lane, & Núñez, 2013), k NH 3 ¼ 4:0 Á 10 8 M −1 S −1 and k NH 3 ¼ 1:1 Á 10 9 M −1 S −1 for T and G, respectively (Moe & Russu, 1992), thus K i X values are readily obtainable from k ex ð½NH 3 Þ dependences.…”

Section: Determination Of Base Pair Opening Temperature (T Bo ) and Mmentioning

confidence: 99%

The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

Kuznetsov

Kiryutin

Кузнецова

et al. 2016

Journal of Biomolecular Structure and Dynamics

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Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, T, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (T(F/T) < T(εA/T) < T(Hx/T) < T(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.

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Base Pair Opening in a Deoxynucleotide Duplex Containing a cis-syn Thymine Cyclobutane Dimer Lesion

Cited by 7 publications

References 59 publications

Enzyme–Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer

Enzyme–Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer

Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle

The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

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