Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent extract of HeLa cells. Synthesis and application of long model DNA

Abstract

Long linear DNA analogs of nucleotide excision repair (NER) substrates have been synthesized. They 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-deoxyguanidine (BP-dG-DNA, 131 bp). 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 of specific excision were shown to vary in the series Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA. During the experiments on affinity modification of the proteins of NER-competent extract, Fap-dC-DNA (137 bp) containing a (32)P-label in the photoactive nucleotide demonstrated properties of a highly efficient and selective probe. The set of the main targets of labeling included polypeptides of the extract with the same values of apparent molecular weights (35-90 kDa) as when using the shorter (48 bp) Fap-dC-DNA. Besides, some of the extract proteins were shown capable of specific and effective interaction with the long analog of NER substrate. Electrophoretic mobility of these proteins coincided with the mobilities of DNA-binding subunits of XPC-HR23B and PARP1 (~127 and ~115 kDa, respectively). The 115-kDa target protein was identified as PARP1 using NAD+-based functional testing. The results suggest that the linear Fap-dC-DNA is an unrepairable substrate analog that can compete with effective NER substrates in the binding of the proteins responsible for lesion recognition and excision.

Molecular Mechanism of Global Genome Nucleotide Excision Repair

“…DNA duplexes of various structures containing bulky modifications, including photoactive fluorochloroazide pyridyl damage, were used as probes. Some model duplexes contained analogs of undamaged strands created with the use of photo reagents with a zero linker length: nucleotide links with 4-thio- and 5-iodo-modified bases [34, 42-44]; some duplexes included a platinum adduct [45]. A large XPC subunit was the only modification target in all cases.…”

“…A large XPC subunit was the only modification target in all cases. The second high-molecular weight nucleoprotein adduct with a lower electrophoretic activity appeared as a result of photo-induced cross-linking with other amino acid residues of the DNA-binding XPC subunit [44]. Moreover, the product of XPC-HR23B proteinprotein cross-links emerging after hard (254 nm) and long-term (60 min) UV irradiation and revealed by Western blotting does not contain a radioactive label and can be formed independently of the presence of a DNA probe [45].…”

Poly(ADP-ribose) Polymerase 1 Modulates Interaction of the Nucleotide Excision Repair Factor XPC-RAD23B with DNA via Poly(ADP-ribosyl)ation

“…Another NER factor, xeroderma pigmentosum protein A also interacts with PARP1 and PAR (10,11). Moreover, PARP1 was identified as one of the main targets in HeLa cell extracts selectively labeled by a photoreactive DNA analogue of the NER substrate (12), suggesting the possible involvement of PARP1 in this repair process. There also exists proteomic data that XPC is one of the protein targets that are (ADP-ribosyl)ated upon induction of genotoxic stress (13,14).…”

New synthetic substrates of mammalian nucleotide excision repair system

“…As a negative control, 137mer DNA strands synthesized with unmodified 16mer ONT were used for duplex formation. The ligation procedure was described in detail elsewhere (20).

Figure 2.The non-nucleoside fragments of the modified DNA strand, containing N -[6-(9-antracenylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol [nAnt, ( A )], N -[6-(5(6)-fluoresceinylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol [nFlu, ( B )] or cholesterol residue [Chol, ( C )].

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“…To synthesize these DNA substrates, the different methods were used, including the chemo-enzymatic approach (2,5,16–19). The improved enzymatic approach for synthesis of bulky substituted DNA structures allowed to synthesize the variety of functional analogues of NER substrates, including the photoactivable ones (20). There is also at least one type of legitimate NER substrate containing bulky non-nucleoside lesions, namely, Chol fragment, introduced by solid-phase DNA synthesis (21).…”