Mechanisms for chromosomal aberrations in mammalian cells

Experimental evidence is presented for the involvement of DNA double-strand breaks in the formation of radiation-induced chromosomal aberrations. When X-irradiated cells were post-treated with single-strand specific Neurospora crassa endonuclease (NE), the frequencies of all classes of aberration increased by about a factor 2. Under these conditions, the frequencies of DNA double-strand breaks induced by X-rays (as determined by neutral sucrose-gradient centrifugation), also increased by a factor of 2. The frequency of chromosomal aberrations induced by fast neutrons (which predominantly induce DNA double-strand breaks) was not influenced by post-treatment with NE. Inhibition of poly(ADP-ribose) polymerase, an enzyme that uses DNA with double-strand breaks as an optimal template, by 3-aminobenzamide also increased the frequencies of X-ray-induced chromosomal aberrations, which supports the idea that DNA double-strand breaks are important lesions for the production of chromosomal aberrations induced by ionizing radiation.     

Activation of poly(adenosine diphosphate ribose) polymerase with UV irradiated and UV endonuclease treated SV 40 minichromosome

SV 40 minichromosomes were used as a molecular model of eukaryotic chromatin to probe the nature of the lesion responsible for UV stimulation of poly (ADPR) polymerase. UV irradiation of the minichromosomes with doses between 50 and 1000 J/m² did not increase their ability to stimulate the activity of purified poly(ADPR) polymerase. In contrast, when the minichromosomes were UV irradiated and then treated with $\text{M.}\text{luteus}$ UV endonuclease, there was a marked increase in their ability to stimulate poly(ADPR) polymerase. This stimulation was completely suppressed when histone Hl was added to the poly(ADPR) polymerase assay. These studies demonstrate in a purified $\text{in}\text{vitro}$ system that damage caused by UV irradiation alone is not sufficient to stimulate poly(ADPR) polymerase activity. Only when DNA is nicked at the site of UV damage by UV endonuclease is there stimulation of poly(ADPR) polymerase.     

Conditions for sliding of nucleosomes along DNA: SV 40 minichromosomes

'Sliding' of nucleosomes along DNA under nearly physiological conditions was studied using treatment of SV 40 minichromosomes with the single-cut restriction endonucleases EcoRI and BamHI. Each enzyme can convert no more than 20-25% of the circular DNA molecules of minichromosomes into the linear form irrespective of the presence of histone H1. This suggests absence of the nucleosomes lateral migration (sliding) along DNa at least in the vicinity of the restriction endonucleases cleavage sites during several hours of incubation. The sites available for EcoRI and BamHI in minichromosomes seem to be located predominantly in the spacer DNA regions of nucleosomes. Introduction of only one double-strand (but not single-strand) break into the DNA of minichromosomes stripped of histone H1 is sufficient to induce redistribution of the nucleosome core particles due to their sliding along DNA. Thus, sliding of the nucleosome core particles can be induced under physiological conditions by rather low energy expenditures.     

Modulation of chromatin structure by poly(ADP-ribosyl)ation

Poly(ADP-ribose) polymerase is a nuclear enzyme that is highly conserved in eucaryotes. Its activity is totally dependent on the presence of DNA containing single or double stranded breaks. We have shown that this activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosy)ation of histone H1. In core particles, the modification of histone H2B leads to a partial dissociation of DNA from core histones. The conformational change of native chromatin by poly(ADP-ribosyl)ation is reversible upon degradation of the histone H1-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. We propose that cuts produced in vivo on DNA during DNA repair activate poly(ADP-ribose) polymerase, which then synthesizes poly(ADP-ribose) on histone H1, in particular, and contributes to the opening of the 25-nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes. This mechanism is fast and reversible.     

Inhibition of DNA damage repair by artificial activation of PARP with siDNA

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.     

Molecular interactions between DNA, poly(ADP-ribose) polymerase, and histones

Molecular interactions between purified poly(ADP-ribose) polymerase, whole thymus histones, histone H1, rat fibroblast genomic DNA, and closed circular and linearized SV40 DNA were determined by the nitrocellulose filter binding technique. Binding of the polymerase protein or histones to DNA was augmented greatly when both the enzyme protein and histones were present simultaneously. The polymerase protein also associated with histones in the absence of DNA. The cooperative or promoted binding of histones and the enzyme to relaxed covalently closed circular SV40 DNA was greater than the binding to the linearized form. Binding of the polymerase to SV40 DNA fragments in the presence of increasing concentrations of NaCl indicated a preferential binding to two restriction fragments as compared to the others. Polymerase binding to covalently closed relaxed SV40 DNA resulted in the induction of superhelicity. The simultaneous influence of the polymerase and histones on DNA topology were more than additive. Topological constraints on DNA induced by poly(ADP-ribose) polymerase were abolished by auto ADP-ribosylation of the enzyme. Benzamide, by inhibiting poly(ADP-ribosylation), reestablished the effect of the polymerase protein on DNA topology. Polymerase binding to in vitro-assembled core particle-like nucleosomes was also demonstrated.     

Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

SV40-3T3 cells were exposed in monolayer cultures to 5 × 10⁻⁷ M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5 × 10⁻⁷ M MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5 × 10⁻⁷ M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle-dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.     

Effects of cell cycle dependent histone H1 phosphorylation on chromatin structure and chromatin replication.

We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M-phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro.     

Poly(ADP-ribosylated) histones in chromatin replication

Poly(ADP-ribosylation) of histones and several other nuclear proteins seem to participate in nuclear processes involving DNA strand breaks like repair, replication, or recombination. This is suggested from the fact that the enzyme poly(ADP-ribose) polymerase responsible for this modification is activated by DNA strand breaks produced in these nuclear processes. In this article I provide three lines of evidence supporting the idea that histone poly(ADP-ribosylation) is involved in chromatin replication. First, cellular lysates from rapidly dividing mouse or human cells in culture synthesize a significant number of oligo- in addition to mono(ADP-ribosylated) histones. Blocking the cells by treatment of cultures with 5 mM butyrate for 24 h or by serum or nutrient depletion results in the synthesis of only mono- but not of oligo(ADP-ribosylated) histones under the same conditions. Thus, the presence of oligo(ADP-ribosylated) histones is related to cell proliferation. Second, cellular lysates or nuclei isolated under mild conditions in the presence of spermine and spermidine and devoid of DNA strand breaks mainly synthesize mono(ADP-ribosylated) histones; introduction of a small number of cuts by DNase I or micrococcal nuclease results in a dramatic increase in the length of poly(ADP-ribose) attached to histones presumably by activation of poly(ADP-ribose) polymerase. Free ends of DNA that could stimulate poly(ADP-ribosylation) of histones are present at the replication fork. Third, putatively acetylated species of histone H4 are more frequently ADP-ribosylated than nonacetylated H4; the number of ADP-ribose groups on histone H4 was found to be equal or exceed by one the number of acetyl groups on this molecule. Since one recognized role of tetraacetylated H4 is its participation in the assembly of new nucleosomes, oligo(ADP-ribosylation) of H4 (and by extension of other histones) may function in new nucleosome formation. Based on these results I propose that poly(ADP-ribosylated) histones are employed for the assembly of histone complexes and their deposition on DNA during replication. Modified histones arise at the replication fork by activation of poly(ADP-ribose) polymerase by unligated Okazaki fragments.     

Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer

The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.     

Regulation of poly(ADP-ribose) polymerase. Histone-specific adaptations of reaction products

The post-translational poly ADP-ribosylation of proteins by the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) involves a complex pattern of ADP-ribose polymers. We have determined how this enzyme produces the various polymer size patterns responsible for altered protein function. The results show that histone H1 and core histones are potent regulators of both the numbers and sizes of ADP-ribose polymers. Each histone induced the polymerase to synthesize a specific polymer size pattern. Various other basic and/or DNA binding proteins as well as other known stimulators of poly(ADP-ribose) polymerase (spermine, MgCl2, nicked DNA) were ineffective as polymer size modulators. Testing specific proteolytic fragments of histone H1, the polymer number and polymer size modulating activity could be mapped to specific polypeptide domains. The results suggest that histones specifically regulate the polymer termination reaction of poly(ADP-ribose) polymerase.     

Poly(ADP-ribose) synthesis and inhibition of DNA synthesis in Chinese hamster cells treated with methylating agents and thymidine

A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [3H]thymidine (dThd) incorporation into DNA and lowering of NAD+ levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 microM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose) synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.     

Time course of polynucleosome relaxation and ADP-ribosylation. Correlation between relaxation and histone H1 hyper-ADP-ribosylation

Isolated rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADP-ribose) polymerase. A time course study was performed using an NAD concentration of 200 microM and changes in nucleosomal structure were investigated by means of electron microscopy visualization and sedimentation velocity determinations. In parallel, analyses of histone H1 poly(ADP-ribosylation) and determinations of DNA polymerase alpha activity on ADP-ribosylated polynucleosomes were done at different time intervals. A direct kinetic correlation between ADP-ribose incorporation, polynucleosome relaxation amd histone H1 hyper-ADP-ribosylation was established. In addition, DNA polymerase alpha activity was highly stimulated on ADP-ribosylated polynucleosomes as compared to control ones, suggesting increased accessibility of DNA to enzymatic action. Because of the strong evidence implicating histone H1 in the maintenance of higher-ordered chromatin structures, the present study may provide a basis for the interpretation of the involvement of the histone H1 ADP-ribosylation reaction in DNA rearrangements during DNA repair, replication or gene expression.     

Poly(ADP-ribose) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

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(ADP0ribose) 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 repairin vivo as a catalyst for nucleosomal unfolding.     

Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA.

The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl-containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA.     

Analysis of DNA double- and single-strand breaks by two dimensional electrophoresis: action of micrococcal nuclease on chromatin and DNA, and degradation in vivo of lens fiber chromatin.

We describe a novel system for two dimensional electrophoresis at neutral and alkaline pH for determining the double-stranded and single-stranded lengths of DNA. With this system we analysed the mode of micrococcal nuclease digestion of DNA in cellular and SV40 viral chromatin and of supercoiled SV40 DNA. The enzyme reaction occurred in two steps : the enzyme first introduced single-strand breaks, then converted these to double-strand breaks by an adjacent cleavage on the opposite strand. Digestion of cellular chromatin DNA occurred by a similar mechanism. Chromatin fragments produced by limited micrococcal nuclease action contained many single-strand breaks, which may be important when this method is used to prepare chromatin fragments for biochemical and biophysical studies. Nucleosome monomer to tetramer produced at later stages of digestion contained few if any single-strand breaks.     

APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells

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(ADP-ribosyl)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.