Base Excision Repair Intermediates as Topoisomerase II Poisons

Amy M Wilstermann,Neil Osheroff

doi:10.1074/jbc.m105733200

Abstract

Abasic sites are the most commonly formed DNA lesions in the cell and are produced by numerous endogenous and environmental insults. In addition, they are generated by the initial step of base excision repair (BER). When located within a topoisomerase II DNA cleavage site, "intact" abasic sites act as topoisomerase II poisons and dramatically stimulate enzyme-mediated DNA scission. However, most abasic sites in cells are not intact. They exist as processed BER intermediates that contain DNA strand breaks proximal to the damaged residue. When strand breaks are located within a topoisomerase II DNA cleavage site, they create suicide substrates that are not religated readily by the enzyme and can generate permanent double-stranded DNA breaks. Consequently, the effects of processed abasic sites on DNA cleavage by human topoisomerase IIalpha were examined. Unlike substrates with intact abasic sites, model BER intermediates containing 5'- or 3'-nicked abasic sites or deoxyribosephosphate flaps were suicide substrates. Furthermore, abasic sites flanked by 5'- or 3'-nicks were potent topoisomerase II poisons, enhancing DNA scission approximately 10-fold compared with corresponding nicked oligonucleotides that lacked abasic sites. These findings suggest that topoisomerase II is able to convert processed BER intermediates to permanent double-stranded DNA breaks.

Highlights

Topoisomerase II is an indispensable enzyme that regulates nucleic acid topology in vivo by passing an intact doublestranded DNA helix through a second segment of DNA
These findings suggest that topoisomerase II is able to convert processed base excision repair (BER) intermediates to permanent doublestranded DNA breaks
Results indicate that when the original lesion was located within the 4-base stagger of a topoisomerase II DNA cleavage site, BER intermediates that contained nicks or DNA flaps acted as suicide substrates for topoisomerase II␣

Summary

Base Excision Repair Intermediates as Topoisomerase II Poisons*

Abasic sites flanked by 5؅- or 3؅nicks were potent topoisomerase II poisons, enhancing DNA scission ϳ10-fold compared with corresponding nicked oligonucleotides that lacked abasic sites These findings suggest that topoisomerase II is able to convert processed BER intermediates to permanent doublestranded DNA breaks. After the creation of abasic sites by the action of the glycosylase, a series of BER intermediates are generated that contain DNA nicks or flaps proximal to the site of the original lesion [32,33,34] Because these intermediates contain strand breaks, they may create substrates that are more lethal for topoisomerase II than the intact abasic site. Results indicate that when the original lesion was located within the 4-base stagger of a topoisomerase II DNA cleavage site, BER intermediates that contained nicks or DNA flaps acted as suicide substrates for topoisomerase II␣.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION