Reconstitution of an in vitro poly(ADP-ribose) turnover system.
Poly(ADP-ribose) is synthesized and degraded by poly(ADP-ribose) polymerase and glycohydrolase, respectively. We have reconstituted in vitro two turnover systems containing these two enzymes. We have measured the kinetics of NAD consumption and polymer accumulation during turnover. The combined action of the two enzymes (i.e., turnover) generates a steady state of polymer quantity. The glycohydrolase determines the time and the level at which this steady state of total polymer is reached. A major observation is that the size and calculated density of polymer bound to the total polymerase molecules is tightly regulated by the rate of polymer turnover. On the polymerase, an increase in the rate of polymer turnover does not affect the mean polymer size, but reduces the polymer density on the enzyme (i.e., the number of polymer chains per polymerase molecule). In the absence of glycohydrolase and at low histone H1 concentration (less than 1.5 micrograms/ml), poly(ADP-ribose) polymerase preferentially automodifies itself instead of modifying histone H1. In contrast, under turnover conditions, oligomer accumulation on histone H1 was greatly increased, with almost 40% of all the polymer present on H1 after 5 min of turnover. Although turnover conditions were necessary for histone H1 labelling, there was no difference between the fast and the slow turnover systems as concerns the proportion of histone H1 labelling, although the mean polymer size on histone H1 was decreased with increasing turnover rate. Due to its small size, polymer is not degraded by the glycohydrolase and accumulates on histone H1 during turnover. These data suggest that the glycohydrolase modulates the level of poly(ADP-ribosyl)action of different proteins in two ways; by degrading shorter polymers at a slower rate and probably by competing with the polymerase for polymer.
Ménard
L
L
Département de Biologie de l'Université de Sherbrooke, Québec.
Thibault
L
L
Poirier
G G
GG
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D006657:Histones / Q000494:pharmacology; D006867:Hydrolases / Q000494:pharmacology; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013950:Thymus Gland / Q000187:drug effects / Q000378:metabolism*
D006657:Histones; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D006867:Hydrolases