From DNA discovery through DNA damage to PAR

In 1869, the Swiss chemist F. Miescher discovered the DNA [1] (Figure 1). After more than eighty years, Watson and Crick made the groundbreaking conclusion that the DNA molecule exists in the form of a three-dimensional double helix. This discovery was based on the analysis of X-ray data of Wilkins and Franklin as well as the Chargaff′s observation that the ratio of adenine to thymine and guanine to cytosine were present in fixed ratios in all studied species [1].

Figure 1 Selected evolutionary landmarks in PARP field. Pictures of scientists taken from: F. Miescher [1], A. Harden [2], F. Crick and J. D. Watson [3], P. Chambon [4], M. Miwa [5], T. Sugimura [6].

At that time, it was not known how many factors are damaging DNA, for example: UV, x-rays, ionizing radiations as well as gamma-rays, viruses, oxidants, alkylating and intercalating agents. Among others, DNA can carry the following damages: oxidation, alkylation, deamination, mismatch of bases,

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single or double strand breaks [7]. With time, more knowledge was acquired about the repair mechanisms such as: reversion repair, mismatch repair (MMR), nucleotide excision repair (NER), DNA double strand brake repair and base excision repair (BER) (Figure 2) [7, 8].

Figure 2 DNA damaging agents, consequences and repair mechanisms. DNA damaging agents cause multiple DNA lesions which are removed and repaired via specific DNA repair pathways. Abbreviations:

cis-Pt: cisplatin and MMC: mitomycin C, respectively (both DNA-crosslinking agents); (6–4) PP:

photoproduct and CPD: 6–4 cyclobutane pyrimidine dimer (both induced by UV light); BER and NER, base- and nucleotide-excision repair, respectively; HR, homologous recombination; EJ, end joining. Figure adapted from [7].

After the discovery of the DNA structure in 1953, the DNA repair involving many proteins has become one of the most interesting topics in modern biology. One of such proteins, which takes part in DNA repair, is poly(ADP-ribose) polymerase (PARP). Investigation of PARP started in the 1960s, when three research groups pioneered the discovery of a novel nucleic acid-like macromolecule, poly(ADP-ribose) PAR. The first group, P. Chambon, J. D. Weill and P. Mandel published in 1963: “We discovered that (…) nicotinamide mononucleotide treatment enhances the activity of a

DNA-15 dependent enzyme which incorporates ATP into a product which is presumably polyadenylic acid; we wish to report some properties of this enzyme which does not seem to have been described as yet” [9]. Initially, it was assumed, that the homopolymeric reaction product is poly-A, but soon there was agreement that the product is indeed PAR [10-13] whose formation is dependent on NAD⁺ which has already been known since 1906 [2]. This finding marked the birth of an intriguing and rapidly growing field of poly(ADP-ribosyl)ation. PAR has aroused the interest of a large number of scientists coming from a very broad range of fields of scientific research.

In 1964, R. J. Collier and A. M. Pappenheimer [14] found that NAD⁺ was required to inhibit the protein synthesis by diphteria toxin in a cell-free system, which led later to the discoveries and to the begin of the research about mono(ADP-ribosyl)ation transferases (MARTs) [15].

M. Miwa and T. Sugimura discovered in 1971 poly(ADP-ribose) glycohydrolase (PARG), an enzyme that cleaves the ribose-ribose bond in poly(ADP-ribose) [16]. Later on, the same authors described in detail the branched structure of poly(ADP-ribose) [16]. The enzyme responsible for the synthesis of poly(ADP-ribose) was named PARP and it was purified to homogenity by many investigators using chromatographic methods. To obtain an electrophoretically pure enzyme, single-step affinity chromatography with 3-aminobenzamide as a ligand was used. Availability of purified PARP allowed to carry out further biochemical studies [17, 18]. However, it took an additional decade to isolate the genes encoding the proteins responsible for ADP-ribosylation reactions. In the late 1980s, the gene encoding a poly(ADP-ribose) synthetase (initially named PARP, ribose) synthetase, or poly(ADP-ribosyl)transferase and now named PARP-1) was isolated [15]. With the cloning of the human PARP cDNA in 1987 by three groups [19-21], the era of molecular biology and molecular genetics of poly(ADP-ribosyl)ation reactions started. The overexpression of cloned PARP cDNAs in various expression systems provided new tools to investigate the modular organization of the enzyme in three functional domains [19, 22].

Easy purification of large amounts of a recombinant chicken PARP catalytic fragment led to the X-ray determination of its structure [23]. In the 80′s,

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the involvement of poly(ADP-ribosyl)ation in DNA repair was proved. The investigations showed that PARP inhibitors such as nicotinamide, benzamide, 3-aminobenzamide prevent rejoining of DNA strand breaks enhanced cytotoxicity even when low dose of alkylating agents was used [24]. This led to the idea of combining DNA-damaging cytotoxic agents used in cancer chemotherapy with PARP inhibitors. PARP has also been shown to be activated independently of DNA strand breakage by binding to DNA structures containing nicks, gaps, cruciforms and DNA bent structures. During the 1970s and 1980s, several laboratories partially purified different enzymes associated with mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation activities. Next, target amino acids for covalent modification of proteins with PAR were identified i.e.

glutamate, aspartate, lysine, and quantitative assays to determine PAR levels in living cells were established [25]. In the 80′s, H. C. Lee and co-workers described one more type of ribosylation reaction: the cyclization of ADP-ribose, which leads to cyclic-ADP-ribose which serves as an important second messenger involved in the regulation of calcium signaling and homeostasis [15].

Recently, the crystal structure of PARP-1 [23, 26, 27] followed by a more comprehensive coverage of the additional members of the PARP family and a histone macrodomain as a PAR binding motif were discovered [28]. Mono(ADP-ribosyl)ation and poly(ADP-Mono(ADP-ribosyl)ation were postulated to be reversible posttranslational modifications of proteins, acting as regulatory mechanisms for proteins.