(5′S) 5′,8-cyclo-2′-deoxyadenosine Cannot Stop BER. Clustered DNA Lesion Studies

Abstract

As a result of external and endocellular physical-chemical factors, every day approximately ~105 DNA lesions might be formed in each human cell. During evolution, living organisms have developed numerous repair systems, of which Base Excision Repair (BER) is the most common. 5′,8-cyclo-2′-deoxyadenosine (cdA) is a tandem lesion that is removed by the Nucleotide Excision Repair (NER) mechanism. Previously, it was assumed that BER machinery was not able to remove (5′S)cdA from the genome. In this study; however, it has been demonstrated that, if (5′S)cdA is a part of a single-stranded clustered DNA lesion, it can be removed from ds-DNA by BER. The above is theoretically possible in two cases: (A) When, during repair, clustered lesions form Okazaki-like fragments; or (B) when the (5′S)cdA moiety is located in the oligonucleotide strand on the 3′-end side of the adjacent DNA damage site, but not when it appears at the opposite 5′-end side. To explain this phenomenon, pure enzymes involved in BER were used (polymerase β (Polβ), a Proliferating Cell Nuclear Antigen (PCNA), and the X-Ray Repair Cross-Complementing Protein 1 (XRCC1)), as well as the Nuclear Extract (NE) from xrs5 cells. It has been found that Polβ can effectively elongate the primer strand in the presence of XRCC1 or PCNA. Moreover, supplementation of the NE from xrs5 cells with Polβ (artificial Polβ overexpression) forced oligonucleotide repair via BER in all the discussed cases.

Effects of 5′,8′-Cyclo-2′-Deoxypurines on the Base Excision Repair of Clustered DNA Lesions in Nuclear Extracts of the XPC Cell Line

“…The results are presented in the Supplementary Materials, i.e., the found and calculated masses are reported in Table S1 and the mass spectra in Figure S2. The substrate oligos were stable in the conditions of the experiment as shown in previous studies: melting temperatures were above 70 • C [42] and matrix strands (native and containing cdPu) were not susceptible to the action of repair enzymes present in NE [2,10,40].…”

“…The two cell lines were used-BJ (human skin fibroblasts) and XPC (skin fibroblasts from Xeroderma Pigmentosum patient, complementation group C, see Section 2.5). The experimental model was, as previously described, synthetic double-stranded oligonucleotides containing dU (AP site precursor) and 5 ,8-cyclo-2deoxyadenosine (cdA) (5 S and 5 R isomer) on the same (ssCDL) or the opposite strand (dsCDL) (Table 1) [10,40,42]. Each step of the substrate ds-oligonucleotide preparation was verified, and the results are presented in Figure S1.…”

“…This leads to the formation of bulky 5 ,8-cyclo-2 -deoxypurines (cdPus) structures in DNA, where the base and sugar moiety of the purine nucleoside in DNA are simultaneously damaged. Therefore, cdPus have been classified as tandem changes which include two adjacent DNA changes located on the same strand [8][9][10]. The production yields of radiation-induced purine 5 ,8-cyclo-2 -deoxyribonucleosides in human monocyte DNA were determined by HPLC-MS/MS of 20 and 4 changes per 10 9 normal nucleosides per Gy for 5 ,8-cyclo-2deoxyguanosine (cdG) and 5 ,8-cyclo-2 -deoxyadenosine (cdA), respectively [11].…”

“…Moreover, differences in BER activity were observed between 5 S and 5 R diastereomers and between purines (cdA and cdG). Repair of the dsCDL was tested in the nuclear extracts (NE) of xrs5 cells, as a well-established model for such experiments [1,10,[37][38][39][40].…”

The Influence of 5′,8-Cyclo-2′-deoxypurines on the Mitochondrial Repair of Clustered DNA Damage in Xrs5 Cells: The Preliminary Study

“…The BER process is impaired when repair enzymes cannot form complexes with DNA due to structural changes in the double helix, such as those resulting from cdPus occurrence [10]. Interestingly, a recent study showed that BER can remove ScdA from the genome, but the lesion location was crucial for proper enzymes' action [26]. Even though the mitochondrial BER (mtBER) is the best-characterized repair system in mitochondria, many questions remain to be answered.…”