High resolution size analysis of ADP-ribose polymers using modified DNA sequencing gels

Panzeter, Phyllis L.; Althaus, Felix R.

doi:10.1093/nar/18.8.2194

Cited by 66 publications

(44 citation statements)

References 2 publications

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“…The position of mADPr ("n"), pADPr of two or more ADPr units (2n, 3n, and 4n, etc. ), and branched pADPr molecules remaining at the origin (16,27) on May 9, 2018 by guest http://mcb.asm.org/ top panels, and B). To examine whether the suppression of pADPr signaling by APLF involved polymer binding, we examined whether the ZNFs were sufficient and/or required for this effect.…”

Section: Vol 28 2008 Aplf and Poly(adp-ribose) Signaling 4623mentioning

confidence: 99%

APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

Rulten

Cortés‐Ledesma

Guo

et al. 2008

Molecular and Cellular Biology

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APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADPribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.The rapid repair of chromosomal DNA single-and doublestrand breaks is critical for genome integrity, and defects in this process result in a variety of hereditary genetic diseases (21). Recently, we and others identified the human protein APLF (aka C2orf13, PALF, and XIP1) as a novel component of the DNA single-strand break repair (SSBR) and double-strand break repair (DSBR) machinery (4,14,15,19). The amino terminus of APLF contains a highly conserved forkhead-associated (FHA) domain that mediates interaction with the SSBR and DSBR factors XRCC1 and XRCC4, respectively. In addition, APLF interacts with Ku80 in an FHA domain-independent manner. The C terminus of APLF contains a second highly conserved region that encodes two tandem zinc fingers (designated ZNF1 and ZNF2) and a highly acidic tail. Both the FHA domain and the tandem ZNFs can facilitate, by independent mechanisms, the accumulation of APLF at sites of DNA strand breakage (4,14,15). Whereas the FHA domain facilitates APLF accumulation via interaction with CK2-phosphorylated XRCC1, the mechanism by which the ZNFs achieve this is unclear.The ZNFs in APLF most closely resemble the tandem zinc fingers present in tristetraprolin. Tristetraprolin binds specific mRNA species, with each of two ZNFs targeting a separate 5Ј-UAUU-3Ј subsite located within a larger, AU-rich recognition sequence (5, 18). Although APLF does not bind this mRNA species (unpublished observations), it is possible that the APLF ZNFs might interact with some other type of adenine-rich structure. One such structure that arises during DNA strand break repair is poly(ADP-ribose) (pADPr), a branched nucleic acid-like polymer synthesized rapidly at DNA strand breaks by pADPr po...

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Section: Vol 28 2008 Aplf and Poly(adp-ribose) Signaling 4623mentioning

confidence: 99%

APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

Rulten

Cortés‐Ledesma

Guo

et al. 2008

Molecular and Cellular Biology

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“…The reaction was stopped by the addition of ice-cold trichloroacetic acid to a final concentration of 20% (w/v). PARs were isolated from trichloroacetic acid precipitates by the method of Panzeter and Althaus (19). Polymers were loaded on a 20% polyacrylamide gel (19:1 acrylamide/bisacrylamide) in 100 mM Tris borate buffer (pH 8.3) and 2 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

PARP-3 Is a Mono-ADP-ribosylase That Activates PARP-1 in the Absence of DNA

Loseva

Jemth

Bryant

et al. 2010

Journal of Biological Chemistry

144

125

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The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADPribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair.Poly(ADP-ribosylation) is a ubiquitous protein modification that is important in the regulation of transcription, cell proliferation, differentiation, and apoptosis. Poly(ADP-ribosylation) occurs in almost all nucleated cells of mammals, plants, and lower eukaryotes but is absent in yeast. It represents an immediate cellular response to DNA damage induced by ionizing radiation, alkylating agents, and oxidants (1, 2). PARP-1 (poly(ADP-ribose) polymerase 1) is an abundant nuclear protein that is activated by DNA strand breaks to modify acceptor proteins with poly(ADP-ribose) (PAR) 4 (3). PARP-1 protects DNA breaks and chromatin structure and recruits DNA repair and checkpoint proteins to sites of damage (4 -6). ADP-ribose is produced from NAD ϩ by cleavage of the glycosidic bond between nicotinamide and ribose, a reaction catalyzed by PARPs. Hydrolysis of the high energy bond between the nicotinamide and ribose produces a free energy of Ϫ34.3 kJ/mol (Ϫ8.2 kcal/mol). This energy is used by PARPs for post-translational modification of proteins by synthesizing ADP-ribose polymers attached to the proteins. PARPs can auto-modify themselves or heteromodify other proteins (1, 2).Human PARPs constitute a large family of 17 proteins encoded by different genes and displaying a conserved catalytic domain. In addition to a catalytic domain, PARP family members typically contain one or more additional motifs or domains, including zinc fingers, BRCT (BRCA1 C terminuslike) motifs, ankyrin repeats, macrodomains and different types of protein/protein interaction sites (7,8). PARP-1 has a highly conserved structure, including an N-terminal DNA-binding domain, a central auto-modification domain, and a C-terminal catalytic domain.Of the human PARP enzymes, at least PARP-1, PARP-2, and tankyrase-1 are required for the maintenance of genome stability. Inhibition of PARP-1 is synthetic lethal with defects in homologous recombination and is currently tested as a monotherapy for heritable breast and...

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“…ADP-ribose polymers of different lengths synthesized by poly(ADP-ribose) polymerase can be separated on DNA sequencing gels and form a unique and distinctive ladder pattern of increasing polymer lengths on the gel (Adamietz et al, 1978;Tanaka et al, 1978;Boulikas, 1990;Keith et al, 1990;Panzeter and Althaus, 1990). The 32P-labelled products synthesized by maize, pea and wheat nuclei were purified and subjected to analysis on 10% polyacrylamide DNA sequencing gels.…”

Section: Gel Electrophoresis Of the Acid-insoluble Radioactivitymentioning

confidence: 99%

Poly(ADP‐Ribose) Polymerase in Plant Nuclei

Chen

Shall

O'Farrell

1994

European Journal of Biochemistry

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We show that poly(ADP-ribose) polymerase is present in maize, pea and wheat nuclei. We have identified the enzyme product as poly(ADP-ribose) by purification and electrophoresis on a DNA sequencing gel. This reveals a polymer ladder consisting of up to 45 residues. The polymer product from maize, after digestion with snake venom phosphodiesterase, gave only 5'-AMP and (phosphoribosy1)-AMP; the mean chain length of the polymer was 5 and 11 residues in two separate experiments. The optimum pH of the plant enzyme is greater than pH 7.0 in pea, wheat and maize; the optimum temperature for enzyme activity is approximately 15 "C. The K, for NAD' for the enzyme from maize is estimated to be approximately 50 pM under optimal conditions. Several compounds (nicotinamide, deoxythymidine, 3-aminobenzamide, 3-methoxybenzamide and 5-bromodeoxyuridine) that specifically inhibit the animal enzyme also inhibit the enzyme from plants. The ratio of the IC,, for 5-bromodeoxyuridine to the IC,, for 3-aminobenzamide in maize is similar to that of the animal enzyme indicating that the enzyme involved is poly(ADP-ribose) polymerase and not mono(ADP-ribosyl) transferase. SDS gel electrophoresis and gel filtration analysis of a crude extract of maize nuclei indicate a molecular mass for poly(ADP-ribose) polymerase of approximately 114 kDa.

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High resolution size analysis of ADP-ribose polymers using modified DNA sequencing gels

Cited by 66 publications

References 2 publications

APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

PARP-3 Is a Mono-ADP-ribosylase That Activates PARP-1 in the Absence of DNA

Poly(ADP‐Ribose) Polymerase in Plant Nuclei

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