(5'S) 5',8-Cyclo-2'-Deoxyadenosine Cannot Stop BER. Clustered DNA Lesion Studies.

Boleslaw T. Karwowski

doi:10.3390/ijms22115934

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.

Highlights

DNA is a miraculously fragile molecule in which the secret code of life can be found
A comparative analysis of the polymerization assay between Nuclear Extract (NE) and NE with XRCC1 or NE/PCNA did not reveal evident differences. These results indicate that scaffold proteins are present in xrs5 NE at the correct/optimal level for other enzymes involved in Base Excision Repair (BER)
The BER pathway was initiated by uracil-DNA glycosylase (UDG), a specific glycosylase that recognizes DNA damage and incises it creating an Apyrimidinic site (AP-site)

Summary

Introduction

DNA is a miraculously fragile molecule in which the secret code of life can be found. It is problematic for small proteins like Polβ or PCNA to keep the template strand in the correct position of the catalytic unit for a subsequent elongation; in the case of dU(+/−), the only negligible dU0 > dU(−)5 > dU(+/−)5 > dU(+)5 > dU(−)3 > dU(+/−)3 > dU(+), which was at the same level as in the previous experiment (Figure 4C) These results indicated the possibility of BER participation in the removal of (5′S)cdA from the genome either when cdPu becomes part of the single‐stranded CDL (Figure 1A) or via the “Okazaki‐like fragment” mecha‐ nism (Figure 1B). It can be postulated that the interaction of XRCC1 with the thumb unit of Polβ stabilized the template DNA strand, which makes the polymerization possible for the widely gapped double helix These results point to and confirm the previous observations (Figure 3B). These observations could become an interesting subject for future painstaking theoretical investigation (Molecular Dynamics)

Conclusions

Materials and Methods