Hanna Kleczkowska - Profile on Academia.edu (original) (raw)
Papers by Hanna Kleczkowska
Additive effects of trifluoperazine or novobiocin and UVC radiation in L5178Y cells
Nukleonika, 1986
Mutation Research/DNA Repair, 1990
The UV254m-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion... more The UV254m-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion into a UV2s4m-resistant, non-tumorigenic LY-S subline after prolonged culture in vitro. Here we describe that this conversion leads to distinct changes in several features of intranuclear ADP-ribose polymer metabolism, which may contribute to the altered processing of DNA damage in these cells. The UV254m-resistant LY-S cells contain 3-fold higher levels of ADP-ribose polymers than LY-R cells. The initial catabolic rate of degradation of these polymers is more than 6-fold higher in LY-S cells. UV254~ m irradiation raises the catabolic rates of ADP-ribose polymers in both cell lines. As a consequence, the polymer half-lives decrease from 15 rain to 4 min in LY-S cells, and from 96 min to 19 min in LY-R cells. In addition, the rapidly turning over fraction of polymers is much larger in the UV254nm-resistant LY-S cells. These data suggest that the catabolism of poly(ADP-ribose) may be an important factor in the biological expression of DNA damage. The involvement of poly(ADP-ribose) metabolism in DNA excision repair has been extensively studied in numerous model systems (for review see
Effect of UVC and araC on L5178Y-R and L5178Y-S cells. Nucleoid sedimentation
Acta biochimica Polonica, 1987
The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine... more The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine (araC, 2 x 10(-3) M, 2 h) on two mouse lymphoma cell lines, UVC-sensitive and X-ray resistant L5178Y-R and UVC-resistant and X-ray sensitive L5178Y-S, were investigated. AraC treatment inhibited the semiconservative DNA replication to 1.4% and 3.8% in L5178Y-R and L5178Y-S cells, respectively, and decreased the sedimentation distance of nucleoids from the cells of both lines. The shortening of sedimentation distances induced by UVC and araC treatment was 8.1 mm for L5178Y-R cells and 11.8 mm for L5178Y-S, and indicated a higher number of DNA breaks in L5178Y-S cells. Assuming that such breaks are the result of the inhibition of DNA repair replication by araC, we conclude that L5178Y-S cells have a greater number of repaired sites than L5178Y-R cells.
Genes & Development, 2001
Proliferating cell nuclear antigen (PCNA) has been implicated in eukaryotic postreplicative misma... more Proliferating cell nuclear antigen (PCNA) has been implicated in eukaryotic postreplicative mismatch correction, but the nature of its interaction with the repair machinery remained enigmatic. We now show that PCNA binds to the human mismatch binding factors hMutSα and hMutSβ via their hMSH6 and hMSH3 subunits, respectively. The N-terminal domains of both proteins contain the highly conserved PCNA-binding motif Qxx[LI]xx[FF]. A variant of hMutSα, lacking this motif because of deletion of 77 N-terminal residues of the hMSH6 subunit, no longer was able to interact with PCNA in vitro and failed to restore mismatch repair in hMSH6-deficient cells. Colocalization of PCNA and hMSH6 or hMSH3 to replication foci implies an intimate link between replication and mismatch correction. We postulate that PCNA plays a role in repair initiation by guiding the mismatch repair proteins to free termini in the newly replicated DNA strands.
Mutation Research/Genetic Toxicology, 1996
Since alkylating agents are widely present in the environment and constitute a continuous challen... more Since alkylating agents are widely present in the environment and constitute a continuous challenge to genome integrity, cells and organisms have developed defense mechanisms to remove such lesions. We monitored the response of human keratinocytes to a very low concentration of a methylating agent, namely 2.5 nM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The effect of a 60-rain exposure of quiescent cells to 2.5 nM MNNG was studied in terms of DNA integrity, poly(ADP-ribose) metabolism, clonogenic survival and DNA synthesis. We observed two waves of DNA strand break formation and resealing. Interestingly, the amount of DNA strand breaks in exposed cells was lower than in unexposed control cells. This phenomenon was also observed when cells were exposed to MNNG in the presence of a protein synthesis inhibitor, or when they were maintained on ice during the treatment. A dose of 2.5 nM MNNG stimulated poly(ADP-ribose) turnover, reduced the intracellular NAD + content, stimulated DNA synthesis and caused a remarkable increase in clonogenic survival. Thus, the cellular responses to extremely low concentrations of MNNG differ sharply from those observed at higher doses of this carcinogen. We conclude that the very low dose response cannot be extrapolated from usual dose-response analyses.
Mutation Research/Genetic Toxicology, 1996
Exposure of cells to low doses of radiation or chemicals renders them more resistant to higher do... more Exposure of cells to low doses of radiation or chemicals renders them more resistant to higher doses of these agents. This phenomenon, termed crdu~~ire r-esporzsr. was studied in quiescent human keratinocytes exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The cells were adapted with 2.5 nM MNNG for 60 min and challenged immediately thereafter with 2.5 PM MNNG for 30, 45 or 60 min. Clonogenic survival studies revealed that adapted cells were more resistant to the subsequent challenge treatment (up to 30% higher survival) than unadapted cells. In addition, formation of DNA strand breaks was lower in adapted cells. We monitored poly-ADP-ribosylation activity during expression of the adaptive response both at the substrate as well as the product level. NAD+ utilization in adapted and non-adapted cells exposed to the high dose of MNNG was similar, but recovery from NAD+ depletion was faster in low-dose pretreated cells. Induction of poly(ADP-ribose) formation was more than 2 times higher in low-dose adapted cells and this was associated with the formation of a distinct class of ADP-ribose polymers, i.e.. branched polymers. These polymers exhibit a very high binding affinity for histones and can displace them from DNA. Elevated levels of poly(kDP-ribosel and, particularly, synthesis of branched polymers may play a critical role in low-dose adaptation.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1996
of the poly(ADP-ribosyl)ation system in the expression of the adaptive response has been demonstr... more of the poly(ADP-ribosyl)ation system in the expression of the adaptive response has been demonstrated with inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase. This enzyme is a key component of a reaction cycle in chromatin, involving dynamic synthesis and degradation of variably sized ADP-ribose polymers in response to DNA strand breaks. The present report reviews recent work focussing on the response of the poly(ADP-ribosyl)ation system in low dose adaptation. The results suggest that adaptation of human cells to minute concentrations of an alkylating agent involves a different activation mechanism for poly(ADP-ribose) polymerase than DNA break-mediated stimulation after high dose treatment. Moreover, adaptation induces the formation of branched polymers with a very high binding affinity for histone tails and selected other proteins. High dose challenge treatment of adapted cells further enhances formation of branched polymers. We propose that apart from sensing DNA nicks, poly(ADP-ribose) polymerase may be part of pathway protecting cells from downstream events of DNA damage.
Adp-Ribosylation and the Response of L5178Y Sublines to Uvc Irradiation
Radiation Research: A Twentieth-century Perspective, 1991
ADP-Ribosylation Reactions: From Bacterial Pathogenesis to Cancer, 1999
Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)p... more Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)polymerase (PARP) does not play a direct role in DNA break processing . Nevertheless, inactivation of the catalytic or the DNA nick-binding functions of PARP affects cellular responses to genotoxins at the level of cell survival, sister chromatid exchanges and apoptosis . In the present report, we conceptualize the idea that PARP is part of a DNA break signal mechanism . In vitro screening studies revealed the existence of a protein family containing a polymer-binding motif of about 22 amino acids. This motif is present in p53 protein as well as in MARCKS, a protein involved in the regulation of the actin cytoskeleton. Biochemical analyses showed that these sequences are directly targeted by PARP-associated polymers in vitro, and this alters several molecular functions of p53-and MARCKS protein. PARP-deficient knockout mice from transgenic mice were found to exhibit several phenotypic features compatible with altered DNA damage signaling, such as downregulation and lack of responsiveness of p53 protein to genotoxins, and morphological changes compatible with MARCKS-related cytoskeletal dysfunction. The knockout phenotype could be rescued by stable expression of the PARP gene. -We propose that PARP-associated polymers may recruit signal proteins to sites of DNA breakage and reprogram their functions. (Mol Cell Biochem 193: 5-11, 1999)
Radiation and Environmental Biophysics, 1988
We examined the response of chromatin to increasing NaC1 and MgC12 concentrations in UVC-irradiat... more We examined the response of chromatin to increasing NaC1 and MgC12 concentrations in UVC-irradiated LS178Y ( L Y ) R and S cells, using the spectral index method . We have found an alteration in chromatin properties 1 h after UVCirradiation of repair proficient LY-S cells, but no change in repair deficient LY-R cells. The change was shown as lowered spectral index, indicating that at given Na + and Mg + + concentrations (1 or 200 mM NaC1, 0 or 0.5 m M MgC12) chromatin from UVC-irradiated LY-S cells was more compact than that from unirradiated ones. Benzamide treatment reversed the effect of UVC-irradiation in LY-S cells and did not change the response pattern of chromatin from LY-R cells or unirradiated LY-S cells.
Molecular and Cellular Biochemistry, 1994
We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(A... more We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(ADP-ribose) glycohydrolase, may cooperate to function as a histone shuttle mechanism on DNA. The mechanism involves four distinct reaction intermediates that were analyzed in a reconstituted in vitro system. In the first step, the enzyme poly(ADP-ribose) polymerase is activated in the presence of histone-DNA complexes and converts itself into a protein carrying multiple ADP-ribose polymers. These polymers attract histones that dissociate from the DNA as a histonepolymer-polymerase complex. The DNA assumes the electrophoretic mobility of free DNA and becomes susceptible to nuclease digestion (second step). In the third step, poly(ADP-ribose) glycohydrolase degrades ADPribose polymers and thereby eliminates the binding sites for histones. In the fourth step, histones reassociate with DNA, and the histone-DNA complexes exhibit the electrophoretic mobilities and nuclease susceptibilities of the original complexes prior to dissociation. Our results are compatible with the view that the poly(ADP-ribosylation) system acts as a catalyst of nucleosomal unfolding of chromatin in DNA excision repair. In DNA excision repair of higher eukaryotes, the processing of NAD+-derived ADP-ribosyl residues by the poly(ADPribosylation) system may be stimulated several thousand-fold. Most of this turnover is associated with the automodification of the nuclear enzyme poly(ADP-ribose) polymerase with multiple ADP-ribose polymers and their rapid degradation by poly(ADP-ribose) glycohydrolase. The biological significance of this automodification cycle is not understood (for reviews, see Ueda and Hayaishi (1985), Althaus and). We have tested the hypothesis that this automodification cycle catalyzes the reversible dissociation of histones from DNA, thereby exposing local DNA domains to other proteins. For this purpose, an in vitro poly(ADP-ribosylation) system was reconstituted in the presence of histone-DNA complexes, and the consequences of poly(ADP-ribosylation) on histone sequestration were analyzed by mobility shift gel electrophoresis and enzyme protection assays. The results show that the two major enzymes responsible for poly(ADP-ribose) turnover in DNA excision repair may catalyze a reaction cycle in which
Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding
Journal of Cellular Biochemistry, 1998
Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due t... more Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system.
Journal of Biological Chemistry, 2000
Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribos... more Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21 CIP1/WAF1 , xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase ⑀, DNA-PK CS , Ku70, NF-B, inducible nitricoxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.
Journal of Biological Chemistry, 1998
Poly ADP-ribosylation in two L5178Y murine lymphoma sublines differentially sensitive to DNA-damaging agents
International Journal of Radiation Biology, 2002
To characterize the response to X-irradiation of the poly ADP-ribosylation system in two closely ... more To characterize the response to X-irradiation of the poly ADP-ribosylation system in two closely related murine lymphoma sublines, L5178Y-R (LY-R) and L5178Y-S (LY-S), with differential sensitivity to various DNA damaging agents (UV-C and ionizing radiation, hydrogen peroxide). LY cells were X-irradiated (2 Gy). NAD+ was determined in cell extracts by high-pressure liquid chromatography. ADP-ribose polymers were purified and analysed by densitometry after polyacrylamide gel electrophoresis. Nuclear matrix proteins were separated by SDS-polyacrylamide gel electrophoresis and processed for ADP-ribose polymer blots to estimate their ability to bind poly(ADP-ribose). In the radiosensitive LY-S cells, the constitutive levels of ADP-ribose polymers were twofold higher than in radioresistant LY-R cells, but unresponsive to a challenge with 2 Gy X-rays. The concentrations of NAD+ - the substrate for poly(ADP-ribose) synthesis - were identical in the two cell lines. X-rays (2 Gy) depleted NAD+ only in LY-S cells. These cells also produced shorter poly(ADP-ribose) molecules as compared with LY-R cells. Nuclear matrix preparations of LY-S cells exhibited lower poly(ADP-ribose)-binding capacity than those of LY-R cells. The results demonstrate disturbances in the poly ADP-ribosylation response of the radiosensitive LY-S cells and reduced poly(ADP-ribose)-binding affinity of the nuclear matrix of these cells.
Environmental and Molecular Mutagenesis, 1993
In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic respon... more In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic response in chromatin. The reaction starts with the binding of a zinc-finger protein, i.e. poly-(ADP-ribose)polymerase to DNA nicks, activation of four resident catalytic activities leading to poly(ADP-ribose) synthesis, conversion of the polymerase into o protein modified with up to 28 variably sized ADP-ribose polymers, and rapid degradation of polymerase-bound polymers by poly(ADP-ribose)glycohydrolase. This auto-~~~~ ~~~~~ modification cycle catalyzes a transient and reversible dissociation of histones from DNA. Shuttling of histones on the DNA allows selected other proteins, such as DNA helicase A and topoisomerase I, to gain access to DNA. Histone shuttling in vitro mimics nucleosomal unfolding/ refolding in vivo that accompanies the postincisional steps of DNA excision repair. Suppression of the automodification cycle in mammalian cells prevents nucleosomal unfolding and nucleotide excision repair.
Cell Biology and Toxicology, 1990
Interactions of poly(ADP-ribose) with nuclear proteins
Biochimie, 1995
The molecular mechanisms whereby poly(ADP-ribosyl)ation primes chromatin proteins for an active r... more 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.
Biochemistry, 1998
In mammalian cells, the formation of DNA strand breaks is accompanied by synthesis of poly(ADP-ri... more In mammalian cells, the formation of DNA strand breaks is accompanied by synthesis of poly(ADP-ribose). This nucleic acid-like homopolymer may modulate protein functions by covalent and/ or noncovalent interactions. Here we show that poly(ADP-ribose) binds strongly to the proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family, MARCKS and MARCKS-related protein (also MacMARCKS or F52). MARCKS proteins are myristoylated proteins associated with membranes and the actin cytoskeleton. As targets for both protein kinase C (PKC) and calmodulin (CaM), MARCKS proteins are thought to mediate cross-talk between these two signal transduction pathways. Dot blot assays show that poly(ADP-ribose) binds to MARCKS proteins at the highly basic effector domain. Complex formation between MARCKS-related protein and CaM as well as phosphorylation of MARCKSrelated protein by the catalytic subunit of PKC are strongly inhibited by equimolar amounts of poly-(ADP-ribose), suggesting a high affinity of poly(ADP-ribose) for MARCKS-related protein. Binding of MARCKS-related protein to membranes is also inhibited by poly(ADP-ribose). Finally, poly(ADP-ribose) efficiently reverses the actin-filament bundling activity of a peptide corresponding to the effector domain and inhibits the formation of actin filaments in Vitro. Our results suggest that MARCKS proteins and actin could be targets of the poly(ADP-ribose) DNA damage signal pathway.
Additive effects of trifluoperazine or novobiocin and UVC radiation in L5178Y cells
Nukleonika, 1986
Mutation Research/DNA Repair, 1990
The UV254m-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion... more The UV254m-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion into a UV2s4m-resistant, non-tumorigenic LY-S subline after prolonged culture in vitro. Here we describe that this conversion leads to distinct changes in several features of intranuclear ADP-ribose polymer metabolism, which may contribute to the altered processing of DNA damage in these cells. The UV254m-resistant LY-S cells contain 3-fold higher levels of ADP-ribose polymers than LY-R cells. The initial catabolic rate of degradation of these polymers is more than 6-fold higher in LY-S cells. UV254~ m irradiation raises the catabolic rates of ADP-ribose polymers in both cell lines. As a consequence, the polymer half-lives decrease from 15 rain to 4 min in LY-S cells, and from 96 min to 19 min in LY-R cells. In addition, the rapidly turning over fraction of polymers is much larger in the UV254nm-resistant LY-S cells. These data suggest that the catabolism of poly(ADP-ribose) may be an important factor in the biological expression of DNA damage. The involvement of poly(ADP-ribose) metabolism in DNA excision repair has been extensively studied in numerous model systems (for review see
Effect of UVC and araC on L5178Y-R and L5178Y-S cells. Nucleoid sedimentation
Acta biochimica Polonica, 1987
The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine... more The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine (araC, 2 x 10(-3) M, 2 h) on two mouse lymphoma cell lines, UVC-sensitive and X-ray resistant L5178Y-R and UVC-resistant and X-ray sensitive L5178Y-S, were investigated. AraC treatment inhibited the semiconservative DNA replication to 1.4% and 3.8% in L5178Y-R and L5178Y-S cells, respectively, and decreased the sedimentation distance of nucleoids from the cells of both lines. The shortening of sedimentation distances induced by UVC and araC treatment was 8.1 mm for L5178Y-R cells and 11.8 mm for L5178Y-S, and indicated a higher number of DNA breaks in L5178Y-S cells. Assuming that such breaks are the result of the inhibition of DNA repair replication by araC, we conclude that L5178Y-S cells have a greater number of repaired sites than L5178Y-R cells.
Genes & Development, 2001
Proliferating cell nuclear antigen (PCNA) has been implicated in eukaryotic postreplicative misma... more Proliferating cell nuclear antigen (PCNA) has been implicated in eukaryotic postreplicative mismatch correction, but the nature of its interaction with the repair machinery remained enigmatic. We now show that PCNA binds to the human mismatch binding factors hMutSα and hMutSβ via their hMSH6 and hMSH3 subunits, respectively. The N-terminal domains of both proteins contain the highly conserved PCNA-binding motif Qxx[LI]xx[FF]. A variant of hMutSα, lacking this motif because of deletion of 77 N-terminal residues of the hMSH6 subunit, no longer was able to interact with PCNA in vitro and failed to restore mismatch repair in hMSH6-deficient cells. Colocalization of PCNA and hMSH6 or hMSH3 to replication foci implies an intimate link between replication and mismatch correction. We postulate that PCNA plays a role in repair initiation by guiding the mismatch repair proteins to free termini in the newly replicated DNA strands.
Mutation Research/Genetic Toxicology, 1996
Since alkylating agents are widely present in the environment and constitute a continuous challen... more Since alkylating agents are widely present in the environment and constitute a continuous challenge to genome integrity, cells and organisms have developed defense mechanisms to remove such lesions. We monitored the response of human keratinocytes to a very low concentration of a methylating agent, namely 2.5 nM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The effect of a 60-rain exposure of quiescent cells to 2.5 nM MNNG was studied in terms of DNA integrity, poly(ADP-ribose) metabolism, clonogenic survival and DNA synthesis. We observed two waves of DNA strand break formation and resealing. Interestingly, the amount of DNA strand breaks in exposed cells was lower than in unexposed control cells. This phenomenon was also observed when cells were exposed to MNNG in the presence of a protein synthesis inhibitor, or when they were maintained on ice during the treatment. A dose of 2.5 nM MNNG stimulated poly(ADP-ribose) turnover, reduced the intracellular NAD + content, stimulated DNA synthesis and caused a remarkable increase in clonogenic survival. Thus, the cellular responses to extremely low concentrations of MNNG differ sharply from those observed at higher doses of this carcinogen. We conclude that the very low dose response cannot be extrapolated from usual dose-response analyses.
Mutation Research/Genetic Toxicology, 1996
Exposure of cells to low doses of radiation or chemicals renders them more resistant to higher do... more Exposure of cells to low doses of radiation or chemicals renders them more resistant to higher doses of these agents. This phenomenon, termed crdu~~ire r-esporzsr. was studied in quiescent human keratinocytes exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The cells were adapted with 2.5 nM MNNG for 60 min and challenged immediately thereafter with 2.5 PM MNNG for 30, 45 or 60 min. Clonogenic survival studies revealed that adapted cells were more resistant to the subsequent challenge treatment (up to 30% higher survival) than unadapted cells. In addition, formation of DNA strand breaks was lower in adapted cells. We monitored poly-ADP-ribosylation activity during expression of the adaptive response both at the substrate as well as the product level. NAD+ utilization in adapted and non-adapted cells exposed to the high dose of MNNG was similar, but recovery from NAD+ depletion was faster in low-dose pretreated cells. Induction of poly(ADP-ribose) formation was more than 2 times higher in low-dose adapted cells and this was associated with the formation of a distinct class of ADP-ribose polymers, i.e.. branched polymers. These polymers exhibit a very high binding affinity for histones and can displace them from DNA. Elevated levels of poly(kDP-ribosel and, particularly, synthesis of branched polymers may play a critical role in low-dose adaptation.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1996
of the poly(ADP-ribosyl)ation system in the expression of the adaptive response has been demonstr... more of the poly(ADP-ribosyl)ation system in the expression of the adaptive response has been demonstrated with inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase. This enzyme is a key component of a reaction cycle in chromatin, involving dynamic synthesis and degradation of variably sized ADP-ribose polymers in response to DNA strand breaks. The present report reviews recent work focussing on the response of the poly(ADP-ribosyl)ation system in low dose adaptation. The results suggest that adaptation of human cells to minute concentrations of an alkylating agent involves a different activation mechanism for poly(ADP-ribose) polymerase than DNA break-mediated stimulation after high dose treatment. Moreover, adaptation induces the formation of branched polymers with a very high binding affinity for histone tails and selected other proteins. High dose challenge treatment of adapted cells further enhances formation of branched polymers. We propose that apart from sensing DNA nicks, poly(ADP-ribose) polymerase may be part of pathway protecting cells from downstream events of DNA damage.
Adp-Ribosylation and the Response of L5178Y Sublines to Uvc Irradiation
Radiation Research: A Twentieth-century Perspective, 1991
ADP-Ribosylation Reactions: From Bacterial Pathogenesis to Cancer, 1999
Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)p... more Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)polymerase (PARP) does not play a direct role in DNA break processing . Nevertheless, inactivation of the catalytic or the DNA nick-binding functions of PARP affects cellular responses to genotoxins at the level of cell survival, sister chromatid exchanges and apoptosis . In the present report, we conceptualize the idea that PARP is part of a DNA break signal mechanism . In vitro screening studies revealed the existence of a protein family containing a polymer-binding motif of about 22 amino acids. This motif is present in p53 protein as well as in MARCKS, a protein involved in the regulation of the actin cytoskeleton. Biochemical analyses showed that these sequences are directly targeted by PARP-associated polymers in vitro, and this alters several molecular functions of p53-and MARCKS protein. PARP-deficient knockout mice from transgenic mice were found to exhibit several phenotypic features compatible with altered DNA damage signaling, such as downregulation and lack of responsiveness of p53 protein to genotoxins, and morphological changes compatible with MARCKS-related cytoskeletal dysfunction. The knockout phenotype could be rescued by stable expression of the PARP gene. -We propose that PARP-associated polymers may recruit signal proteins to sites of DNA breakage and reprogram their functions. (Mol Cell Biochem 193: 5-11, 1999)
Radiation and Environmental Biophysics, 1988
We examined the response of chromatin to increasing NaC1 and MgC12 concentrations in UVC-irradiat... more We examined the response of chromatin to increasing NaC1 and MgC12 concentrations in UVC-irradiated LS178Y ( L Y ) R and S cells, using the spectral index method . We have found an alteration in chromatin properties 1 h after UVCirradiation of repair proficient LY-S cells, but no change in repair deficient LY-R cells. The change was shown as lowered spectral index, indicating that at given Na + and Mg + + concentrations (1 or 200 mM NaC1, 0 or 0.5 m M MgC12) chromatin from UVC-irradiated LY-S cells was more compact than that from unirradiated ones. Benzamide treatment reversed the effect of UVC-irradiation in LY-S cells and did not change the response pattern of chromatin from LY-R cells or unirradiated LY-S cells.
Molecular and Cellular Biochemistry, 1994
We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(A... more We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(ADP-ribose) glycohydrolase, may cooperate to function as a histone shuttle mechanism on DNA. The mechanism involves four distinct reaction intermediates that were analyzed in a reconstituted in vitro system. In the first step, the enzyme poly(ADP-ribose) polymerase is activated in the presence of histone-DNA complexes and converts itself into a protein carrying multiple ADP-ribose polymers. These polymers attract histones that dissociate from the DNA as a histonepolymer-polymerase complex. The DNA assumes the electrophoretic mobility of free DNA and becomes susceptible to nuclease digestion (second step). In the third step, poly(ADP-ribose) glycohydrolase degrades ADPribose polymers and thereby eliminates the binding sites for histones. In the fourth step, histones reassociate with DNA, and the histone-DNA complexes exhibit the electrophoretic mobilities and nuclease susceptibilities of the original complexes prior to dissociation. Our results are compatible with the view that the poly(ADP-ribosylation) system acts as a catalyst of nucleosomal unfolding of chromatin in DNA excision repair. In DNA excision repair of higher eukaryotes, the processing of NAD+-derived ADP-ribosyl residues by the poly(ADPribosylation) system may be stimulated several thousand-fold. Most of this turnover is associated with the automodification of the nuclear enzyme poly(ADP-ribose) polymerase with multiple ADP-ribose polymers and their rapid degradation by poly(ADP-ribose) glycohydrolase. The biological significance of this automodification cycle is not understood (for reviews, see Ueda and Hayaishi (1985), Althaus and). We have tested the hypothesis that this automodification cycle catalyzes the reversible dissociation of histones from DNA, thereby exposing local DNA domains to other proteins. For this purpose, an in vitro poly(ADP-ribosylation) system was reconstituted in the presence of histone-DNA complexes, and the consequences of poly(ADP-ribosylation) on histone sequestration were analyzed by mobility shift gel electrophoresis and enzyme protection assays. The results show that the two major enzymes responsible for poly(ADP-ribose) turnover in DNA excision repair may catalyze a reaction cycle in which
Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding
Journal of Cellular Biochemistry, 1998
Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due t... more Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system.
Journal of Biological Chemistry, 2000
Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribos... more Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21 CIP1/WAF1 , xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase ⑀, DNA-PK CS , Ku70, NF-B, inducible nitricoxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.
Journal of Biological Chemistry, 1998
Poly ADP-ribosylation in two L5178Y murine lymphoma sublines differentially sensitive to DNA-damaging agents
International Journal of Radiation Biology, 2002
To characterize the response to X-irradiation of the poly ADP-ribosylation system in two closely ... more To characterize the response to X-irradiation of the poly ADP-ribosylation system in two closely related murine lymphoma sublines, L5178Y-R (LY-R) and L5178Y-S (LY-S), with differential sensitivity to various DNA damaging agents (UV-C and ionizing radiation, hydrogen peroxide). LY cells were X-irradiated (2 Gy). NAD+ was determined in cell extracts by high-pressure liquid chromatography. ADP-ribose polymers were purified and analysed by densitometry after polyacrylamide gel electrophoresis. Nuclear matrix proteins were separated by SDS-polyacrylamide gel electrophoresis and processed for ADP-ribose polymer blots to estimate their ability to bind poly(ADP-ribose). In the radiosensitive LY-S cells, the constitutive levels of ADP-ribose polymers were twofold higher than in radioresistant LY-R cells, but unresponsive to a challenge with 2 Gy X-rays. The concentrations of NAD+ - the substrate for poly(ADP-ribose) synthesis - were identical in the two cell lines. X-rays (2 Gy) depleted NAD+ only in LY-S cells. These cells also produced shorter poly(ADP-ribose) molecules as compared with LY-R cells. Nuclear matrix preparations of LY-S cells exhibited lower poly(ADP-ribose)-binding capacity than those of LY-R cells. The results demonstrate disturbances in the poly ADP-ribosylation response of the radiosensitive LY-S cells and reduced poly(ADP-ribose)-binding affinity of the nuclear matrix of these cells.
Environmental and Molecular Mutagenesis, 1993
In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic respon... more In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic response in chromatin. The reaction starts with the binding of a zinc-finger protein, i.e. poly-(ADP-ribose)polymerase to DNA nicks, activation of four resident catalytic activities leading to poly(ADP-ribose) synthesis, conversion of the polymerase into o protein modified with up to 28 variably sized ADP-ribose polymers, and rapid degradation of polymerase-bound polymers by poly(ADP-ribose)glycohydrolase. This auto-~~~~ ~~~~~ modification cycle catalyzes a transient and reversible dissociation of histones from DNA. Shuttling of histones on the DNA allows selected other proteins, such as DNA helicase A and topoisomerase I, to gain access to DNA. Histone shuttling in vitro mimics nucleosomal unfolding/ refolding in vivo that accompanies the postincisional steps of DNA excision repair. Suppression of the automodification cycle in mammalian cells prevents nucleosomal unfolding and nucleotide excision repair.
Cell Biology and Toxicology, 1990
Interactions of poly(ADP-ribose) with nuclear proteins
Biochimie, 1995
The molecular mechanisms whereby poly(ADP-ribosyl)ation primes chromatin proteins for an active r... more 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.
Biochemistry, 1998
In mammalian cells, the formation of DNA strand breaks is accompanied by synthesis of poly(ADP-ri... more In mammalian cells, the formation of DNA strand breaks is accompanied by synthesis of poly(ADP-ribose). This nucleic acid-like homopolymer may modulate protein functions by covalent and/ or noncovalent interactions. Here we show that poly(ADP-ribose) binds strongly to the proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family, MARCKS and MARCKS-related protein (also MacMARCKS or F52). MARCKS proteins are myristoylated proteins associated with membranes and the actin cytoskeleton. As targets for both protein kinase C (PKC) and calmodulin (CaM), MARCKS proteins are thought to mediate cross-talk between these two signal transduction pathways. Dot blot assays show that poly(ADP-ribose) binds to MARCKS proteins at the highly basic effector domain. Complex formation between MARCKS-related protein and CaM as well as phosphorylation of MARCKSrelated protein by the catalytic subunit of PKC are strongly inhibited by equimolar amounts of poly-(ADP-ribose), suggesting a high affinity of poly(ADP-ribose) for MARCKS-related protein. Binding of MARCKS-related protein to membranes is also inhibited by poly(ADP-ribose). Finally, poly(ADP-ribose) efficiently reverses the actin-filament bundling activity of a peptide corresponding to the effector domain and inhibits the formation of actin filaments in Vitro. Our results suggest that MARCKS proteins and actin could be targets of the poly(ADP-ribose) DNA damage signal pathway.