Coenzymatic Activity of Randomly Broken or Intact Double-stranded DNAs in Auto and Histone H1 Trans-poly(ADP-ribosylation), Catalyzed by Poly(ADP-ribose) Polymerase (PARP I)

Abstract

The enzymatic transfer of ADP-ribose from NAD to histone H 1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V max . Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K a ‫؍‬ 1400 versus 3-15, respectively). Mg 2؉ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg 2؉ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADPribosylation), thereby opening the possibility to study the physiologic function of PARP I.The activity of poly(ADP-ribose) polymerase (PARP I, 1 EC 2.4.2.30) was originally considered to represent a novel form of covalent protein modification analogous to phosphorylation, acetylation, etc. (1). Immunochemical analysis of normal adult rat tissues for poly(ADP-ribose) (pADPR) showed that 99% of covalently bound polymer was found in the non-histone fraction of nuclear proteins, with histone H 1 being the major acceptor among the remaining 1% (2). The actual quantities of protein-bound pADPR in normal tissues, however, are small (200 -250 ng/g, dry weight) as compared with the large abundance of PARP I protein itself (0.5 ϫ 10 6 copies/cell (cf. Ref. 4). This difference suggests that PARP I activity in vivo is highly regulated. The secondary structure of pADPR (3) predicts that binding of modified proteins to other proteins or to DNA will be altered by poly(ADP-ribosylation). Furthermore, unmodified PARP I avidly binds to a large number of nuclear proteins (4), an association that is completely blocked by added histone H 1 . In addition, an assortment of transcription factors was shown to bind to PARP I (cf. Ref. 5), and a transcriptional regulatory role of PARP I in muscle differentiation has been identified (6). The MCAT element derived from this work (cf. Ref. 6) served as a basic DNA sequence for the construction of dsDNAs studied in the present experiment. Recently the topology of the direct binding of PARP I to Topo 1 has been demonstrated in detail (7). The binding of PARP I to histone H 1 induces conformational changes in the latter coinciding with new phosphorylation sites for Cdc2-kinase (8).Although the enzymatic transfer of ADP-ribose from NAD to PARP I and to several nuclear proteins has been well established (1), it has remained unclear what factors determine protein auto-or heteromodification by poly(ADP-ribos...

In vivo regulation of the chicken cardiac troponin T gene promoter in zebrafish embryos

“…2.4.2.30; Butler and Ordahl, 1999) is a chromatin associated, nuclear enzyme that modifies itself and other nuclear proteins, such as histone H1, through addition of poly-(ADP-ribose) (Kun et al, 2002). Because the mt4 mutation disrupts PARP-I binding (Butler and Ordahl, 1999), the results presented here support the hypothesis that PARP-I-dependent repression/derepression regulation, as seen in vitro by transfection of cultured chick embryo cells, functions similarly in vivo.…”

“…Historically, PARP-I has been most extensively studied in cell death pathology, in part because of the widely held belief that only "broken" or "damaged" DNA serves as its coenzyme (deMurcia and Shall, 2000). The recent demonstration that double-strand DNA is a more efficient coenzyme for PARP-I than does DNA with strand breaks (Kun et al, 2002), provides a physiological basis for its role in gene regulation. In addition, PARP-IЈs DNA-bindingdependent enzymatic modification of other proteins substantially expands its potential regulatory effects on transcription (Mendoza-Alvarez and Alvarez-Gonzalez, 2001;Oei and Shi, 2001a, b).…”

Mechanism of early biphasic activation of poly(ADP-ribose) polymerase-1 in response to ultraviolet B radiation

“…We have already shown in the in vitro assays that PARP can bind to and be activated by UVB-irradiated DNA in the absence of damage processing repair enzymes. Moreover, PARP is known to be activated by unusual DNA structures without DNA strand breaks, such as cruciform, curved and bent structures (Rolli et al, 2000) or linear and stem-loop structures (Kun et al, 2002). Furthermore, PARP activation and chromatin loosening in the absence of DNA strand breaks has also been observed during transcriptional activation of genes in Drosophila salivary gland chromosome (Tulin and Spradling, 2003).…”

Modulation of DNMT1 activity by ADP-ribose polymers

“…In normal cells, the unmodified PARP1 molecules considerably outnumber the poly(ADP-ribosyl)ated ones (D'Amours et al, 1999;Kun et al, 2002). This situation can be explained considering that PARP1 is involved both in situations of cellular emergency (D'Amours et al, 1999;Ame´et al, 2000) and in physiologic regulation of biological events (Kraus and Lis, 2003).…”