Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone H1 trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I).
The enzymatic transfer of ADP-ribose from NAD to histone H1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V(max). Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K(a) = 1400 versus 3-15, respectively). Mg2+ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg2+ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADP- ribosylation), thereby opening the possibility to study the physiologic function of PARP I.
Department of Anatomy, San Francisco School of Medicine, University of California, San Francisco, California 94143, USA.
Ernest
E
Kun
Eva
E
Kirsten
Charles P
CP
Ordahl
D000244:Adenosine Diphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006657:Histones / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology
D018076:DNA, Complementary; D006657:Histones; D000244:Adenosine Diphosphate; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D008274:Magnesium