PARENT SESSION
3:45 PM to 5:15 PM
Monday, April 22, 2002
Mini-Symposium 8
DNA Repair
Room: Nevada 6-7
, Co-Chair: Kao, Gary¹; Glazer, Peter²¹University of Pennsylvania, Philadelphia, PA²Yale University, New Haven, CT

(MS08-1) Detecting clustured DNA abasic sites by use of polyamines.

Georgakilas, Alexandros^*,1, Bennett, Paula¹, Sutherland, Betsy¹, ¹ Biology Department, Brookhaven National Laboratory, Upton, NY

ABSTRACT-
There is accumulating evidence that the repair of clustered DNA damage induced by ionizing radiation might be impaired by the insufficient cleavage of endonucleases and other repair enzymes participating in the recognition and response of the cell to DNA damage. A key class of clustered damage is the abasic cluster−two or more closely spaced abasic sites (AP) on opposing strands−analyzed by cleavage with Escherichia coli Endonuclease IV (Sutherland et al., 2000, Proc. Natl. Acad. Sci. USA, 97:103). However, Chaudry and Weinfeld showed that Ape1, which also recognizes abasic sites, does not cleave at all such clusters (1997, J. Biol. Chem., 275:15650). Polyamines (putrescine, spermine and spermidine) can detect and cleave abasic sites (AP) introduced in DNA by several damaging agents. Thus, it seemed likely that polyamines might cleave abasic clusters more efficiently than Endonuclease IV. We tested this hypothesis by using polyamines to cleave T7 bacteriophage -irradiated DNA (0-10 Gy) as well as 23-mer oligonucleotide duplexes containing abasic sites at specific lesions either directly opposite each other or 1, 3 or 5 nucleotides apart in the 5^' or 3^' direction. We found that Endonuclease IV had varying activity on the oligonucleotides substrates used, depending on the relative position of the two abasic sites. However, putrescine was remarkably efficient in cleaving all the clusters tested. We found for T7 DNA, that putrescine is the most efficient cleavage agent and shows substantially higher efficiency compared with Endonuclease IV. This research was supported by the Office of Biological and Environmental Research of the Department of Energy, and by a grant R01 CA86897 from the National Institutes of Health to BMS.

KEYWORDS: Abasic sites, Putrescine, EndonucleaseIV, Ionizing radiation