Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair

Abstract

IntroductionThe apurinic/apyrimidinic (AP) site is considered to be a common lesion in genomic DNA, arising at a frequency of 10,000 to 50,000 lesions per mammalian cell per day [1]. The number of AP sites can increase dramatically under stressful conditions such as X-ray or UV light irradiation and alkylating agent exposure. If unrepaired, AP sites present mutagenic and cytotoxic consequences to the cell [2]. The loss of DNA bases and attendant formation of AP sites in DNA occurs spontaneously as a result of hydrolytic cleavage of N-glycosylic bonds or through glycosylasecatalyzed removal of damaged bases during the early stage of base excision repair (BER) [3]. The steady-state levels of AP sites in mammalian cells are in the range of approximately 1 site per 10 6 nucleotides [4]. Repair of AP sites is thought to be extremely rapid [5]. At the same time, it has been found that a part of AP sites can persist in genomic DNA for a rather long period [6]. This observation raised a question about nature of these slowly repaired AP sites and the mechanisms of prompt and dilatory repair. It is widely accepted that, irrespectively of their origin, AP sites are further processed by the BER machinery. In most cases, the repair of AP sites is initiated by hydrolytic cleavage 5′ to an AP site catalyzed by apurinic/apyrimidinic endonuclease 1 (APE1) [7]. AP sites also can be incised through β-elimination Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair via the activity of DNA glycosylases and other enzymes with associated AP lyase activity [3,7]. In this case, strand incision occurs 3′ to an AP site, generating α,β-unsaturated aldehyde (α,β-4-hydroxypenten-2-al) at the 3′-margin and phosphate at the 5′-margin, and this intermediate is further processed by the downstream BER enzymes [7]. This scenario appears to be realized in the case of isolated AP sites, which are generated under moderate level of DNA damage. But under some highly stressful conditions, e.g. exposure to ionizing radiation, AP sites can be a part of multiply damaged sites containing also oxidized bases, and single-or double-strand breaks (SSBs or DSBs) [8,9]. Indeed, among additional types of damage produced by high linear energy transfer irradiation, AP sites, within 8-10 bp of a DS end, were found to be most frequent [10]. the following steps. First, the preparative BHT of cell extract proteins with 32 P-labelled AP DNA, which contains a biotin moiety, is performed. Second, the cross-linked protein is purified by affinity chromatography on streptavidin-coated paramagnetic beads and resolved by SDS-PAGE. A well-defined Coomassie stained protein band precisely corresponding to the radioactive label is excised from the gel, and the protein is subjected to ingel trypsin digestion and MALDI-TOF-MS. Results from the MS analyses are searched against a database to identify the crosslinked protein. It should be pointed out that in this approach AP sites play two roles, the naturally occurring DNA lesion an...

Coordination of DNA Base Excision Repair by Protein-Protein Interactions

“…PARP1 forms stable complexes with Ku70/Ku80 proteins, and this interaction has been proposed to be an important regulator of the Ku70/80 heterodimer function in the repair of DNA double-strand breaks (DSBs) [91,92]. Recent studies have demonstrated the involvement of Ku70 and Ku80 proteins in different steps of BER [93]. Septin4, a member of GTP binding protein family considered to be an essential component of the cytoskeleton, is a novel PARP1 interacting protein, and the interaction is enhanced under oxidative stress [94].…”