Hilda Mendoza-Alvarez

Enzymology of ADP-ribose polymer synthesis

In this minireview, we summarize recent advances on the enzymology of ADP-ribose polymer synthesis. First, a short discussion of the primary structure and cloning of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], the enzyme that catalyzes, the synthesis of poly(ADP-ribose), is presented. A catalytic distinction between the multiple enzymatic activities of PARP is established. The direction of ADP-ribose chain growth as well as the molecular mechanism of the automodification reaction catalyzed by PARP are described. Current approaches to dissect ADP-ribose polymer synthesis into individual reactions of initiation, elongation and branching, as well as a partial mechanistic characterization of the ADP-ribose elongation reaction at he chemical level are also presented. Finally, recent developments in the catalytic characterization of PARP by site-directed mutagensis are also briefly summarized.

Regulatory mechanisms of poly(ADP-ribose) polymerase.

Here, we describe the latest developments on the mechanistic characterization of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], a DNA-dependent enzyme that catalyzes the synthesis of protein-bound ADP-ribose polymers in eucaryotic chromatin. A detailed kinetic analysis of the automodification reaction of PARP in the presence of nicked dsDNA indicates that protein-poly(ADP-ribosyl)ation probably occurs via a sequential mechanism since enzyme-bound ADP-ribose chains are not reaction intermediates. The multiple enzymatic activities catalyzed by PARP (initiation, elongation, branching and self-modification) are the subject of a very complex regulatory mechanism that may involve allosterism. For instance, while the NAD+ concentration determines the average ADP-ribose polymer size (polymerization reaction), the frequency of DNA strand breaks determines the total number of ADP-ribose chains synthesized (initiation reaction). A general discussion of some of the mechanisms that regulate these multiple catalytic activities of PARP is presented below.

Functional Interactions of PARP-1 with p53

A close correlation between the frequency of specific mutations of oncogenes and/or tumor suppressor genes in mammals and cancer has been suspected for a long time. For instance, either spontaneous or forcefully inflicted mutations of a tumor suppressor gene coding for a protein known as p53 are usually associated with a variety of malignant tumors. Overwhelming experimental evidence indicates that more than 50% of human neoplasias1 contain one or multiple mutations in one or both alleles of p53. Therefore, the expression product of this pivotal gene, when mutated, appears to play a major role in carcinogenesis. Further significance of p53, as a tumor suppressor protein, is underscored by the fact that over 90% of all tumor-derived mutations associated with it, result in structural and/or functional alterations of its sequence-specific DNA-binding domain2 (Seq-Sp DBD, Fig. 1). Interestingly, the high mutation frequency observed with p53 in malignant tissues initially lead to the misidentification of a mutant of this chromosomal locus as an oncogene rather than its wild type version which functions as the opposite, a tumor suppressor product. An overwhelming amount of work has been done in the last few years to unveil the physiological, biochemical and molecular significance of p53, especially at the protein level. However, in this review the discussion centers on the relevance of the p53 structure and function relationships with poly(ADP-ribose) polymerase-1 (PARP-1), a prominent DNA-strand break sensor in higher eucaryotes, and the biochemical pathway of protein-poly(ADP-ribosyl)ation. A special emphasirity. Therefore, we will present the primary sequence and domain structure of both proteins first.

Chain Length Analysis of ADP‐Ribose Polymers Generated by Poly(ADP‐Ribose) Polymerase (PARP) as a Function of ß‐NAD and Enzyme Concentrations

Bireactant autopoly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30) was carried out by using either increasing concentrations of beta-NAD+ (donor substrate) at a fixed protein concentration or increasing concentrations of PARP (acceptor substrate) at a fixed beta-NAD+ concentration. The [32P]ADP-ribose polymers synthesized were chemically detached from PARP by alkaline hydrolysis of the monoester bond between the carboxylate moiety of Glu and the polymer. Nucleic acid-like polymers were then analyzed by high-resolution polyacrylamide gel electrophoresis and autoradiography. The ADP-ribose chain lengths observed displayed substrate concentration-dependent elongation from 0.2 microM to 2 mM beta-NAD+. Similar results were observed at fixed concentrations of 4.5, 9, 18, 27, and 36 nM PARP. Therefore, we conclude that the concentration of the ADP-ribose donor substrate determines the average chain length of the polymer synthesized. In contrast, the polymer size was unaltered when the concentration of PARP was varied from 4.5 to 18 nM at a fixed beta-NAD+ concentration. However, when PARP concentrations > 18 nM were used, the total amount of monomeric ADP-ribose produced was noticeably less. Therefore, we conclude that high concentrations of PARP lead to acceptor substrate inhibition at the level of the ADP-ribose chain initiation reaction.

Poly (3’-deoxyADP-ribosyl)ation of Proteins in Liver Chromatin Isolated from Rats Fed with Hepatocarcinogens

We have recently found that 3’-deoxyNAD is a good substrate for poly(ADP-ribose)polymerase (PADPRP) (1,2). In fact, we observed that PADPRP makes small linear oligomers of 3’-deoxyADP-ribose with an average size of 4 ADP-ribose residues. The main advantage of this approach is that the highly branched and complex polymers of ADP-ribose synthesized with NAD (3) are not observed. Therefore, the electrophoretic identification of poly(ADPribosyl)ated-polypeptides following incubation of biological samples possessing PADPRP activity with [32P] 3’-deoxyNAD is facilitated.

Up-Regulation of Two Distinct p53-DNA Binding Functions by Covalent Poly(ADP-ribosyl)ation: Transactivating and Single Strand Break Sensing

We used a [32P] p53 sequence-specific oligodeoxynucleotide and Electrophoretic-Mobility-Shift-Assays to monitor p53 DNA sequence-specific binding with p53-R267W, a nonbinding point mutant; and p53-Δ30, a deletion-mutant which lacks the carboxy-terminus that recognizes DNA-strand-breaks. Recombinant p53 and poly(ADP-ribose)polymerase-1 (PARP-1) were incubated with labeled βNAD+ with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and βNAD+ and p53 concentration(s). Since p53-Δ30 was efficiently labeled, poly(ADP-ribosyl)ation target site(s) of wt-p53 must reside outside its carboxy-terminal-domain. The poly(ADP-ribosyl)ation of p53-Δ30 did not diminish its DNA binding; Instead, it enhanced DNA-sequence-specific-binding. Therefore, we conclude that DNA-sequence-specific-binding and DNA-nick-sensing of mutant-p53 are differentially regulated by poly(ADP-ribosyl)ation.