Poly(ADP-ribose) synthesis in vitro programmed by damaged DNA. A comparison of DNA molecules containing different types of strand breaks

Abstract

The ability of DNA is support poly(ADP-ribose) synthesis is completely dependent upon the number and type of strand breaks it contains and is independent of the sequence. Single-stranded DNA is ineffective. Covalently closed circular plasmid DNA is ineffective, but when it is enzymatically digested it activates poly(ADP-ribose) polymerase in proportion to the number of strand breaks, suggesting that the polymerase recognizes DNA ends. Double-stranded restriction fragments with flush ends are approximately 3 times more effective than are fragments with unpaired nucleotides extending from the 3' termini and about 10 times more effective than are either fragments with unpaired nucleotides extending from the 5' termini or plasmids with single-strand breaks. All types of restriction fragments become more effective upon removal of terminal 5'-phosphate groups. This specificity profile may relate to the proposed role of poly(ADP-ribose) synthesis in the repair of DNA strand breaks, for those which are assumed to be more difficult to repair in vivo are the more effective stimulators. Poly(ADP-ribose) polymerase has no divalent cation requirement when supplied with flush-ended DNA fragments, but magnesium may enhance the effective of other types of DNA by activating magnesium-dependent nucleases. Ineffective DNAs, such as covalently closed plasmids or synthetic homopolymers that are unable to form Watson-Crick duplexes, apparently compete with effective DNA and weakly inhibit poly(ADP-ribose) synthesis.