Poly ADP-ribosylation in two L5178Y murine lymphoma sublines differentially sensitive to DNA-damaging agents.

PURPOSE: 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). MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.

hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication foci.

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 hMutSalpha and hMutSbeta 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 hMutSalpha, 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.

Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins.

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 epsilon, DNA-PK(CS), Ku70, NF-kappaB, inducible nitric-oxide 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.

Poly ADP-ribosylation: a DNA break signal mechanism.

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.

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding.

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.

Poly(ADP-ribose) modulates the properties of MARCKS proteins.

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 MARCKS-related 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.

Poly(ADP-ribose) binds to specific domains of p53 and alters its DNA binding functions.

DNA strand breaks are potential interaction sites for the nuclear enzyme poly(ADP-ribose) polymerase (PARP; E.C. 2.4.2.30) and the tumor suppressor protein p53. Both proteins bind and respond to DNA breaks and both play a role in DNA damage signaling. A temporary colocalization and complex formation between these proteins has been demonstrated in mammalian cells. Here we show that free and poly(ADP-ribose) polymerase-bound ADP-ribose polymers target three domains in p53 protein for strong noncovalent interactions. The polymer binding sites could be mapped to two amino acid sequences in the sequence-specific core DNA binding domain of p53 (amino acid positions 153-178 and 231-253) and another one in the oligomerization domain (amino acids 326-348). In mobility shift experiments, poly(ADP-ribose) effectively prevented and reversed p53 binding to the palindromic p53 consensus sequence. Additionally, poly(ADP-ribose) also interfered with the DNA single strand end binding of p53. The results suggest that ADP-ribose polymers could play a role in regulating the DNA binding properties of p53.

The role of poly(ADP-ribosyl)ation in the adaptive response.

An involvement 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.

Biochemical changes associated with the adaptive response of human keratinocytes to N-methyl-N'-nitro-N-nitrosoguanidine.

Exposure of cells to low doses of radiation or chemicals renders them more resistant to higher doses of these agents. This phenomenon, termed adaptive response, 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 microM 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(ADP-ribose) and, particularly, synthesis of branched polymers may play a critical role in low-dose adaptation.

Response of human keratinocytes to extremely low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine.

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-min 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.

Interactions of poly(ADP-ribose) with nuclear proteins.

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.

Histone shuttling by poly ADP-ribosylation.

The enzymes poly(ADP-ribose)polymerase and poly(ADP-ribose) glycohydrolase may cooperate to drive a histone shuttle mechanism in chromatin. The mechanism is triggered by binding of the N-terminal zinc-finger domain of the polymerase to DNA strand breaks, which activates the catalytic activities residing in the C-terminal domain. The polymerase converts into a protein carrying multiple ADP-ribose polymers which displace histones from DNA by specifically targeting the histone tails responsible for DNA condensation. As a result, the domains surrounding DNA strand breaks become accessible to other proteins. Poly(ADP-ribose)glycohydrolase attacks ADP-ribose polymers in a specific order and thereby releases histones for reassociation with DNA. Increasing evidence from different model systems suggests that histone shuttling participates in DNA repair in vivo as a catalyst for nucleosomal unfolding.

Histone shuttle driven by the automodification cycle of poly(ADP-ribose)polymerase.

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 a protein modified with up to 28 variably sized ADP-ribose polymers, and rapid degradation of polymerase-bound polymers by poly(ADP-ribose)glycohydrolase. This automodification 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.

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells.

We have determined the ability of UV254nm-irradiated murine lymphoma cells to adapt their NAD+ metabolism to the increased NAD+ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD(+)-utilizing enzyme poly(ADP-ribose) polymerase, NAD+ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV254nm irradiation, a dramatic transient increase of NAD+ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV254nm irradiation, the NAD+ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD+ metabolism to UV254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD+ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells.

Differential poly(ADP-ribose) metabolism in repair-proficient and repair-deficient murine lymphoma cells.

The UV254nm-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion into a UV254nm-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 UV254nm-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. UV254nm irradiation raises the catabolic rates of ADP-ribose polymers in both cell lines. As a consequence, the polymer half-lives decrease from 15 min 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.

Benzamide-inhibitable alterations in spectral properties of chromatin accompany DNA repair in UVC-exposed L5178Y cells.

We examined the response of chromatin to increasing NaCl and MgCl2 concentrations in UVC-irradiated L5178Y (LY) R and S cells, using the spectral index method (Dixon and Burkholder 1985). We have found an alteration in chromatin properties 1 h after UVC-irradiation 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 NaCl, 0 or 0.5 mM MgCl2) 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.