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

R.C Benjamin,D.M Gill

doi:10.1016/s0021-9258(19)70491-8

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.

Highlights

The ability of DNA to support poly(ADP-ribose)syn- thymus ( 5 ) .Caplan showed, that nucleotide sequence thesis is completely dependent upon the number and was probably of little importance, for unique and repeated type of strand breaks it contains and is independentof sequenceDNAfragments of thesameaverage size were the sequence
(16).There is usually sufficient endogenousDNA to saturate the enzyme andthe addition of DNA generally produceslittle or no increase inthe rate of poly(ADP-ribose)synthesis. Passage of this high salt extract of calf thymus overa DEAE-cellulose columnseparatesafraction of the poly(ADP-ribose) polymerase that is almost completely dependent on exogeneous DNA for activity (Fig. l).The rate of polymer synthesis is increasedmorethan40-foldby the addition of 1 pg/ml of pBR322 digested with Hae I11 endonuclease
Nature of the Product-Polyacrylamide gel analysis in the orthat supercoiling preventstheDNA from stimulating presence of sodium dodecyl sulfate reveals that the NAD’- poly(ADP-ribose) synthesis.Upon the introduction of one or derived product is principally composed of free polymers of two single-strand breaks by limited DNase I digestion, the ADP-ribose, the majority being over 50 residues in length, DNA becomes able to support poly(ADP-ribose) synthesis

Summary

RESULTS

Thymus-Asreportedearlier,a 0.5 M NaClextractofcalf thymus contains both DNA fragments and poly(ADP-ribose) polymerase. Passage of this high salt extract of calf thymus overa DEAE-cellulose columnseparatesafraction of the poly(ADP-ribose) polymerase that is almost completely dependent on exogeneous DNA for activity (Fig. l).The rate of polymer synthesis is increasedmorethan40-foldby the addition of 1 pg/ml of pBR322 digested with Hae I11 endonuclease This DNA-stimulated activity has a K,,, for NAD'. A significant result is thatpBR322 supercoils, even a t high concentrations, are totally unable to support poly(ADP-ribose) synthesis (Fig. 4) This is the first ineffective double-stranded DNA to be found. Nature of the Product-Polyacrylamide gel analysis in the orthat supercoiling preventstheDNA from stimulating presence of sodium dodecyl sulfate reveals that the NAD’- poly(ADP-ribose) synthesis.Upon the introduction of one or derived product is principally composed of free polymers of two single-strand breaks by limited DNase I digestion, the ADP-ribose, the majority being over 50 residues in length, DNA becomes able to support poly(ADP-ribose) synthesis.

DNA added

DISCUSSION

Effect of MagnesiuIomonns