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DNA with damage in both strands as affinity probes and nucleotide excision repair substrates

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Abstract

Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dCFAB and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dCFAB/dG (probe I), dCFAB/nFlu+4 (probe II), and dCFAB/nFlu−3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC—HR23B (Kd um > Kd I > Kd II ≈ Kd III ) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dCFAB results in (i) decreased melting temperature (ΔTm = −3°C) and (ii) 12° DNA bending. The extended dCFAB/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC—HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dCFAB/nFlu+4, dCFAB/nFlu−3) or irreparable (nFlu/nFlu+4, nFlu/nFlu−3, nAnt/nFlu+4, nAnt/nFlu−3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis.

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Abbreviations

dCFAB :

5-[3-(4-azido-2,3,5,6-tetrafluorobenzamido)propoxyprop-1-inyl]-2′-deoxycytidine

dCFABG :

exo-N-[(4-azi-dotetrafluorobenzylidenehydrazinocarbonyl)butylcarbamoyl]-2′-deoxycytidine

dCFAP :

exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]amino-ethyl}-2′-deoxycytidine

dsDNA:

double-stranded DNA

dUFAP :

5-{N-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2′-deoxy-uridine

Kd:

dissociation constant

n:

nucleotide

nAnt:

a nonnucleoside insert containing anthracenyl residue (N-[6-(9-anthracenyl-carbamoyl)hexanoyl]-3-amino-1,2-propanediol)

NER:

nucleotide excision repair

nFlu:

a nonnucleoside insert containing fluorescein residue (N-[6-(dipivaloyl-5(6)-fluoresceinyl-carbamoyl)hexanoyl]-O1-(4,4′-dimethoxytrityl)-O2-[(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1,2-propanediol)

ODN:

oligo(deoxy)ribonucleotide

RPA:

replication protein A

TFIIH:

transcription factor II human

Tm:

DNA duplex melting temperature

T4 PNK:

T4 polynucleotide kinase

umDNA:

unmodified DNA

XPC and XPD:

xeroderma pigmentosum group C

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Authors and Affiliations

  1. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090, Novosibirsk, Russia

    N. V. Lukyanchikova, I. O. Petruseva, A. N. Evdokimov, V. N. Silnikov & O. I. Lavrik

  2. Novosibirsk State University, 630090, Novosibirsk, Russia

    N. V. Lukyanchikova & O. I. Lavrik

  3. Altai State University, 656049, Barnaul, Russia

    O. I. Lavrik

Authors
  1. N. V. Lukyanchikova
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  2. I. O. Petruseva
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  3. A. N. Evdokimov
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  4. V. N. Silnikov
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  5. O. I. Lavrik
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Correspondence to O. I. Lavrik.

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Original Russian Text © N. V. Lukyanchikova, I. O. Petruseva, A. N. Evdokimov, V. N. Silnikov, O. I. Lavrik, 2016, published in Biokhimiya, 2016, Vol. 81, No. 3, pp. 386–400.

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Lukyanchikova, N.V., Petruseva, I.O., Evdokimov, A.N. et al. DNA with damage in both strands as affinity probes and nucleotide excision repair substrates. Biochemistry Moscow 81, 263–274 (2016). https://doi.org/10.1134/S0006297916030093

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  • DOI: https://doi.org/10.1134/S0006297916030093

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