Antibody to poly(adenosine diphosphate-ribose) polymerase and its use in chromfltin analysis

Abstract

To facilitate investigations on the organization of poly (ADP-Rib) polymerase in chromatin, and to elucidate its biological function, polymerase purified from HeLa nuclei was used to elicit antibodies in mice. The anti-polymerase sera was found to be specific by multiple criteria. The association of polymerase with oligonucleosomes of differing chain size was determined by the specific binding of polymerase antibody (and as control, anti-histone H3) to nitrocellulose transfers of native electrophoretic gels of these particles. By this technique, polymerase appears to be associated with a select subclass of nucleosomes. Further resolution of the polymerase binding sites within nucleosome classes was achieved with the antibody by two-dimensional native polyacrylamide gel electrophoresis and nitrocellulose transfer.

Immunoaffinity fractionation of the poly(ADP-ribosyl)ated domains of chromatin.

“…4). It has been reported (7,9) that, under these labeling conditions, incorporated 32p migrates with polymerase-active oligonucleosomal species. The unbound nucleosomes contained the majority of the nucleosomal chromatin applied to the column, yet no polymerase activity remained with these classes of chromatin (lane 2).…”

“…Accordingly, oligonucleosomes were incubated in the presence and absence of 10 AuM NAD, subsequently separated by nondenaturating polyacrylamide gel electrophoresis, blotted (9), and allowed to react with anti-poly(ADP-ribose). Significant binding of the IgG to each chain size of nucleosomes was noted in the chromatin sample preincubated with NAD (data not shown).…”

“…1) (14)(15)(16). The automodified polymerase has been shown by various criteria to be reasonably tightly bound to the internucleosomal region of chromatin (7,10,(13)(14)(15), and antibody to the polymerase has been shown to react with blots of electrophoretically separated nucleosomes (9).…”

Immunological evidence for the in vivo occurrence of a crosslinked complex of poly(ADP-ribosylated) histone H1.

“…[32P]NAD for 30 sec, after which Hi was selectively extracted and subjected to electrophoresis and autoradiography (lane 1).…”

Cloning and expression of cDNA for human poly(ADP-ribose) polymerase.

“…The antibody used in the screening was a rabbit antiserum against a pure preparation of HeLa cell poly(ADP-ribose) polymerase (9). A similar antibody has been described (10).…”