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

Abstract

Clustered DNA lesions (CDL) containing 5′,8-cyclo-2′-deoxypurines (cdPus) are an example of extensive abnormalities occurring in the DNA helix and may impede cellular repair processes. The changes in the efficiency of nuclear base excision repair (BER) were investigated using (a) two cell lines, one of the normal skin fibroblasts as a reference (BJ) and the second from Xeroderma pigmentosum patients’ skin (XPC), and (b) synthetic oligonucleotides with single- and double-stranded CDL (containing 5′,8-cyclo-2′-deoxyadenosine (cdA) and the abasic (AP) site at various distances between lesions). The nuclear BER has been observed and the effect of both cdA isomers (5′R and 5′S) presence in the DNA was tested. CdPus affected the repair of the second lesion within the CDL. The BER system more efficiently processed damage in the vicinity of the ScdA isomer and changes located in the 3′-end direction for dsCDL and in the 5′-end direction for ssCDL. The presented study is the very first investigation of the repair processes of the CDL containing cdPu considering cells derived from a Xeroderma pigmentosum patient.

Mutagenicity of a bi-stranded clustered DNA lesion containing (5’S) or (5’R) 5’,8-cyclo-2’-deoxyAdenosine in Escherichia coli model

“…2). This observation was in good agreement with our previous, recently published studies of complex CDL in which RcdA was more easily repaired than ScdA by nuclear and mitochondrial extracts from eukaryotic cells (Boguszewska et al, 2021a;Kaźmierczak-Barańska et al, 2021;Boguszewska et al, 2021b). RcdA bends the DNA double helix to a greater extent than ScdA; therefore, DNA repair enzymes are more likely to recognize and remove it from the plasmid sequence (Karwowski, 2013).…”

“…The mutation frequency increased in the following order: RcdA/dU0<ScdA/dU0<RcdA/dU-1<ScdA/ dU-1<RcdA/dU+1<ScdA/dU+1. The fact that clusters containing dU+1 (for both diastereomers of cdA) have the highest mutagenic potential corresponded to previous studies in eukaryotic models (Pearson et al, 2004;Cunniffe et al, 2014;Karwowski, 2019;Boguszewska et al, 2021a;Kaźmierczak-Barańska et al, 2021;Boguszewska et al, 2021b;Karwowski et al, 2014). It was found that the most abundant mutation in the case of ScdA/dU+1 was dU:G deletion (30.8%).…”

“…Subsequently, cdPus may interfere with repair and replication in Escherichia coli because in most cases polymerases cannot bypass its complex structure and Nucleotide Excision Repair (NER) is inefficient (Jasti et al, 2011;David-Cordonniert et al, 2000;Pednekar et al, 2014). When CDL contains an apurinic/apyrimidinic site (AP site) and cdPu, the efficiency of proteins involved in the repair of the AP site through the Base Excision Repair (BER) machinery can decrease, as shown in our previous studies on nuclear and mitochondrial extracts of eukaryotic cells (Karwowski, 2019;Boguszewska et al, 2021a;Kaźmierczak-Barańska et al, 2021;Boguszewska et al, 2021b;Karwowski et al, 2014;Karwowski, 2021). The AP site located opposite to 5′,8-cyclo-2′-deoxyAdenosine (cdA), or 1 nucleobase in the 5′-end direction was found to be a cause of the termination of ds-oligo repair, as polymerases cannot operate properly.…”

“…The DNA repair capacity differed depending on the type of cdPu, its diastereomeric form (5′S or 5′R), and the interlesion distance. These features directly influenced the activity of proteins involved in DNA repair (Karwowski, 2019;Boguszewska et al, 2021a;Szewczuk et al, 2021;Kaźmierczak-Barańska et al, 2021;Boguszewska et al, 2021b). When eukaryotic DNA repair enzymes cannot form active complexes with DNA due to structural changes (e.g., resulting from cdPu occurrence), the DNA lesion remains in the cell unre- paired, which can lead to mutations and finally to pathogenic states (e.g., carcinogenesis and/or accelerated aging).…”

The Usefulness of Autoradiography for DNA Repair Proteins Activity Detection in the Cytoplasm towards Radiolabeled Oligonucleotides Containing 5′,8-Cyclo-2′-deoxyadenosine

“…For example, AP sites are usually cleaved before base damage when located 5 bp from each other, and the resulting SSB is repaired first [28]. Furthermore, studies have shown that the presence of cdA impacts the action of repair proteins and/or the DNA repair efficiency of the second lesion within the cluster [7,8,10,19,[29][30][31][32][33].…”