Biopolym. Cell. 2012; 28(3):212-217.
Structure and Function of Biopolymers
Synthesis of model DNA and their application as substrates of nucleotide excision repair
1Evdokimov A. N., 1, 2Tsidulko A. Yu., 1Petruseva I. O., 1, 2Koroleva L. S., 1Serpokrylova I. Yu., 1Silnikov V. N., 1, 2Lavrik O. I.
  1. Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
    8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090
  2. Novosibirsk State University
    2, Pirogova Str., Novosibirsk, Russian Federation, 630090

Abstract

Aim. Nucleotide excision repair (NER) is DNA repair system responsible to remove bulky lesions from DNA. These lesions appear in DNA as consequence of UV-light irradiation or environmental stress. Study of NER is extremely important to improve action of chemotherapeutic drugs. Methods. In vitro NER-assay and photoaffinity modification were used. Results. Long linear DNA analogs mimicking NER substrates have been synthesized. DNA analogs are 137-mer duplexes containing in their internal positions nucleotides with bulky substitutes imitating lesions with fluorochloroazidopyridyl and fluorescein groups introduced using spacer fragments at the 4N and 5C positions of dCMP and dUMP (Fap-dC- and Flu-dU-DNA) and DNA containing a (+)-cis-stereoisomer of benzo[a]pyrene-N2-deoxyguanosine (BP-dG-DNA). The interaction of the modified DNA duplexes with the proteins of NER-competent HeLa extract was investigated. The substrate properties of the model DNA in the reaction ofspecific excision were shown to vary in the row Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA. Conclusions. In vitro assay show that DNA analogs represent an interesting tool for the estimation of cellular repair activities. The developed approach should be of general use forthe incorporation of NER-sensitive distortions into model DNA and seems to be very promising for repair mechanism studies.
Keywords: nucleotide excision repair, model bulky substituted DNA substrates

References

[1] Scharer O. D. Chemistry and biology of DNA repair Angew. Chem. Int. Ed. Engl 2003 42, N 26:2946–2974.
[2] Dip R., Camenisch U., Naegeli H. Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair DNA Repair (Amst) 2004 3, N 11:1409–1423.
[3] Gillet L. C., Scharer O. D. Molecular mechanisms of mammalian global genome nucleotide excision repair Chem. Rev 2006 106, N 2:253–276.
[4] Sugasawa K., Okamoto T., Shimizu Y., Masutani C., Iwai S., Hanaoka F. A multistep damage recognition mechanism for global genomic nucleotide excision repair Genes Dev 2001 15, N 5:507–521.
[5] Hey T., Lipps G., Sugasawa K., Iwai S., Hanaoka F., Krauss G. The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay Biochemistry 2002 41, N 21:6583–6587.
[6] Tapias A., Auriol J., Forget D., Enzlin J. H., Scharer O. D., Coin F., Coulombe B., Egly J. M. Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors J. Biol. Chem 2004 279, N 18:19074–19083.
[7] DellaVecchia M. J., Croteau D. L., Skorvaga M., Dezhurov S. V., Lavrik O. I., Van Houten B. Analyzing the handoff of DNA from UvrA to UvrB utilizing DNA-protein photoaffinity labeling J. Biol. Chem 2004 279, N 43:45245–45256.
[8] Trego K. S., Turchi J. J. Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination Biochemistry 2006 45, N 6:1961–1969.
[9] Huang J. C., Sancar A. Determination of minimum substrate size for human excinuclease J. Biol. Chem 1994 269, N 29 P. 19034–19040.
[10] Reardon J. T., Sancar A. Purification and characterization of Escherichia coli and human nucleotide excision repair enzyme systems Methods Enzymol 2006 408:189–213.
[11] Buterin T., Meyer C., Giese B., Naegeli H. DNA quality control by conformational readout on the undamaged strand of the double helix Chem. Biol 2005 12, N 8:913–922.
[12] Sugasawa K., Akagi J., Nishi R., Iwai S., Hanaoka F. Two-step recognition of DNA damage for mammalian nucleotide excision repair: directional binding of the XPC complex and DNA strand scanning Mol. Cell 2009 36, N 4:642–653.
[13] Mocquet V., Kropachev K., Kolbanovskiy M., Kolbanovskiy A., Tapias A., Cai Y., Broyde S., Geacintov N. E., Egly J. M. The human DNA repair factor XPC-HR23B distinguishes stereoisomeric benzo[a]pyrenyl-DNA lesions EMBO J 2007 26, N 12 P. 2923–2932.
[14] Kropachev K., Kolbanovskii M., Cai Y., Rodriguez F., Kolbanovskii A., Liu Y., Zhang L., Amin S., Patel D., Broyde S., Geacintov N. E. The sequence dependence of human nucleotide excision repair efficiencies of benzo[a]pyrene-derived DNA lesions: insights into the structural factors that favor dual incisions J. Mol. Biol 2009 386, N 5:1193–1203.
[15] Dezhurov S. V., Khodyreva S. N., Plekhanova E. S., Lavrik O. I. A new highly efficient photoreactive analogue of dCTP. Synthesis, characterization, and application in photoaffinity modification of DNA binding proteins Bioconjug. Chem 2005 16, N 1:215–222.
[16] Nishi R., Okuda Y., Watanabe E., Mori T., Iwai S., Masutani C., Sugasawa K., Hanaoka F. Centrin 2 stimulates nucleotide excision repair by interacting with xeroderma pigmentosum group C protein Mol. Cell. Biol 2005 25, N 13:5664–5674.
[17] Evdokimov A. N., Petruseva I. O., Pestryakov P. E., Lavrik O. I. Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NERcompetent extract of HeLa cells. Synthesis and application of long model DNA Biochemistry (Mosc) 2011 76, N 1:157–166.
[18] Petruseva I. O., Tikhanovich I. S., Chelobanov B. P., Lavrik O. I. RPA repair recognition of DNA containing pyrimidines bearing bulky adducts J. Mol. Recognit 2008 21, N 3:154–162.
[19] Petruseva I. O., Tikhanovich I. S., Maltseva E. A., Safronov I. V., Lavrik O. I. Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent HeLa cell extract Biochemistry (Mosc) 2009 74, N 5:491–501.
[20] Buterin T., Hess M. T., Gunz D., Geacintov N. E., Mullenders L. H., Naegeli H. Trapping of DNA nucleotide excision repair factors by nonrepairable carcinogen adducts Cancer Res 2002 62, N 15:4229–4235.