Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity

Abstract

The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks

“…PARP1 dimerisation at sites of DNA damage has been implicated in multiple previous studies of PARP1 activation 1,7,[26][27][28] , although this has not been universally observed 20,21 .…”

“…A substantial body of data suggest that PARP1 becomes activated at sites of DNA damage by trans-modification of one PARP1 molecule by another, with formation at least transiently, of a dimeric interaction between DNA-bound PARP1 molecules 1,7,[26][27][28] . Two recent studies 20,21 , while observing DNA binding modes consistent with dimerization or oligomerization in some circumstances, were able to define buffer conditions and DNA molecules in which 1:1 protein:DNA complexes predominated.…”

A Third Zinc-binding Domain of Human Poly(ADP-ribose) Polymerase-1 Coordinates DNA-dependent Enzyme Activation

“…PARP-1 self-association is linked to maximal DNAdependent enzyme activation (12), and several regions of PARP-1 are known to form interdomain contacts (12,41,42,46). Kinetic analysis of PARP-1 activity and the stoichiometry of PARP-1 binding to DNA indicate that PARP-1 is a catalytic dimer (22,37).…”

“…DNA strand breaks stimulate PARP-1 automodification, and activated PARP-1 recruits DNA repair factors to the site of DNA damage to facilitate repair (10,11). There are several factors that control PARP-1 activity, including self-association, interaction with histones and nucleosomes, NAD ϩ concentrations, structure-specific binding to DNA, and automodification (1)(2)(3)12). However, there are few molecular level insights into mechanisms that control PARP-1 activity.…”

Evidence for the participation of histidine residues located in the 56 kDa C‐terminal polypeptide domain of ADP‐ribosyl transferase in its catalytic activity

“…It is of interest that DEP reacts with only two histidine residues of the 56 kDa polypeptide but not with imidazole molecules present in the 29 kDa polypeptide domain. This apparent selectivity is unexplained, unless one considers macromolecular structural factors such as dimerization [18] that may determine this selectivity towards DEP under given experimental conditions. Our results thus provide preliminary evidence for the localization of the catalytic site of ADPRT in the 56 kDa carboxy-terminal polypeptide domain, coinciding with a previous suggestion [19], which is based on specific thiol labelling of ADPT.…”