Phyllis L. Panzeter

HISTONE SHUTTLING BY POLY ADP-RIBOSYLATION

The enzymes poly(ADP-ribose)polymerase and poly(ADP-ribose) glycohydrolase may cooperate to drive a histone shuttle mechanism in chromatin. The mechanism is triggered by binding of the N-terminal zinc-finger domain of the polymerase to DNA strand breaks, which activates the catalytic activities residing in the C-terminal domain. The polymerase converts into a protein carrying multiple ADP-ribose polymers which displace histones from DNA by specifically targeting the histone tails responsible for DNA condensation. As a result, the domains surrounding DNA strand breaks become accessible to other proteins. Poly(ADP0ribose) glycohydrolase attacks ADP-ribose polymers in a specific order and thereby releases histones for reassociation with DNA. Increasing evidence from different model systems suggests that histone shuttling participates in DNA repairin vivo as a catalyst for nucleosomal unfolding.

Interactions of poly(ADP-ribose) with nuclear proteins

The molecular mechanisms whereby poly(ADP-ribosyl)ation primes chromatin proteins for an active role in DNA excision repair are not understood. The prevalent view is that the covalent linkage of ADP-ribose polymers is essential for the modification of target protein function. By contrast, we have focused on the possibility that ADP-ribose polymers interact non-covalently with nuclear proteins and thereby modulate their function. The results show that ADP-ribose polymers engage in highly specific and strong non-covalent interactions with a small number of nuclear proteins, predominantly histones, and among these only with specific polypeptide domains. The binding affinities were largely determined by two factors, ie the polymer sizes and the presence of branches. This provides an explanation for the target specificity of the histone shuttle mechanism that was previously reported by our laboratory. Interestingly, the polymer molecules being most effective in protein targeting in vitro, are strictly regulated in mammalian cells during DNA repair in vivo.

Synthesis of poly(ADP-robose)-agarose beads: An affinity resin for studying (ADP-ribose)n-protein interactions

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.

Fast protein liquid chromatographic purification of poly(ADP-ribose) polymerase and separation of ADP-ribose polymers

Abstract Poly(ADP-ribose) polymerase responds to DNA strand breaks in nuclei by producing ADP-ribose polymers covalently attached to proteins. Here we report two fast protein liquid chromatographic applications to aid investigations on poly(ADP-ribosyl)ation. The first rapidly purifies poly(ADP-ribose) polymerase from crude calf thymus extract. The purification protocol, involving successive fractionations over four columns, reduces the time for polymerase purification from four days to 14 h resulting in a > 50% increase in enzyme-specific activity. The second application employs a complex salt gradient to reproducibly separate ADP-ribose polymers into individual size classes.

The Poly(ADP-ribose)-Protein Shuttle of Chromatin

Since the discovery of poly(ADP-ribose) in Paul Mandel’s laboratory in 196E (1), poly ADP-ribosylation has been classified as a posttranslational protein modification (for review see 2-4), While this definition is formally correct, it may not precisely reflect the primary function of poly(ADP-ribose). The term “posttranslational modification” projects the idea that the modifying residue modifies protein function by covalent modification of the target acceptor. Over the past five years, we have focussed on the possibility that ADP-ribose polymers may primarily act on protein function(s) by non-covalent interactions with nuclear proteins. The working hypothesis envisions poly(ADP-ribose) metabolism as a protein shuttle mechanism in chromatin. The results show that poly ADP-ribosylation may selectively regulate DNA template accessibility for proteins involved in DNA processing functions. In this mechanism, poly(ADP-ribose) assumes the function of an alternative polynucleotide binding site for histones in chromatin. Testing this concept in models of various complexities, we conclude that protein shuttling by poly ADP-ribosylation may be involved in local unfolding of nucleosomes during DNA excision repair (5,6).

The α-glycosidic bonds of poly(ADP-ribose) are acid-labile

Abstract The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The α-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.

ADP-ribose Polymers Bind Specifically and Non-covalently to Histones

Poly ADP-ribosylation of nuclear proteins may induce alterations in chromatin structure which affect chromatin function (for reviews see 4, 2). Covalent, post-translational modification of histones by poly(ADP-ribose) has been particularly investigated as the most probable mode of influencing DNA structure (3-6). However, under DNA damage conditions, the enzyme responsible for polymer formation, poly(ADP-ribose) polymerase, is itself the most highly modified protein in vivo (7). An apparent paradox of post-translational protein modification arises: the modifying enzyme becomes the predominant, modified protein. If’ the main function of poly(ADP-ribose) polymerase is to modify itself, what then is the function of modified poly(ADP-ribose) polymerase? We propose that the polymers covalently bound to poly(ADP-ribose) polymerase themselves influence chromatin structure by non-covalently interacting with chromatin proteins, i.e. histones (8). This paper describes the non-covalent interactions between histones and poly(ADP-ribose) in vitra