RPA repair recognition of DNA containing pyrimidines bearing bulky adducts.
Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.
Petruseva
Irina O
IO
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva, 8, Novosibirsk 630090, Russia.
Tikhanovich
Irina S
IS
Chelobanov
Boris P
BP
Lavrik
Olga I
OI
D001483:Base Sequence; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000378:metabolism; D004260:DNA Repair; D005454:Fluorescence Polarization; D006801:Humans; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D019920:Photoaffinity Labels; D011743:Pyrimidines / Q000737:chemistry*; D051716:Replication Protein A / Q000378:metabolism*
D018736:DNA Adducts; D015342:DNA Probes; D009711:Nucleotides; D019920:Photoaffinity Labels; D011743:Pyrimidines; D051716:Replication Protein A; C030986:pyrimidine