Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid and mouse melanoma cells

DNA endonuclease activities from nuclear proteins of normal human and xeroderma pigmentosum (XP), complementation group A, lymphoblastoid and Cloudman mouse melanoma cells were examined against partially apurinic/apyrimidinic (AP) DNA. Non-histone chromatin-associated and nucleoplasmic proteins, obtained from isolated nuclei, were subfractionated by isoelectric focusing and assayed for DNA endonuclease activity against linear, calf thymus DNA. All of the nine chromatin-associated and three of the nucleoplasmic fractions, which lacked DNA exonuclease activity, were tested for DNA endonuclease activity against both native and partially AP, circular, duplex, supercoiled PM2 DNA. In all three cell lines, four chromatin-associated, but none of the nucleoplasmic fractions, showed increased activity against DNA rendered AP by either heat/acid treatment or by alkylation with methyl methanesulfonate (MMS) followed by heat. One chromatin-associated activity, with pI 9.8, which was not active on native DNA, showed the greatest activity on AP DNA. AP activity was moderately decreased in XP cells and slightly decreased in mouse melanoma cells, as compared with normal cells, in the fraction at pI 9.8. Little or no increased activity was observed in any of the endonucleases from any of the cell lines on MMS alkylated DNA.     

DNA glycosylases of human lymphoblastoid cells.

Lymphoblastoid cell lines were established after transformation by Epstein-Barr virus of peripheral lymphocytes from xeroderma pigmentosum (XP) patients and normal donors. These lines expressed B-lymphocyte characteristics. Typical characteristics related to XP of these cell lines were not altered by transformation. Extracts of these cells catalyzed release of uracil (Ura) and 3-methyladenine (3MeAde) from Ura-containing DNA (Ura-DNA) and methylated DNA (Me-DNA), respectively. These two activities, Ura-DNA glycosylase and 3MeAde-DNA glycosylase, differed in heat stability. Extracts released Ura more rapidly and 3MeAde more slowly from a single-stranded DNA than from a double-stranded DNA. On incubation with reconstituted chromatins prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein, cell extracts released all the Ura but about half the 3MeAde residues. The activity levels of these two enzymes of XP cells were similar to those of normal cells.     

Repair of damaged DNA by extracts from a xeroderma pigmentosum complementation group A revertant and expression of a protein absent in its parental cell line.

Cells derived from individuals with mutations in the xeroderma pigmentosum complementation group A gene (XP-A gene) are hypersensitive to UV light and have a severe defect in nucleotide excision repair of damaged DNA. UV-resistant revertant cell lines can arise from XP-A cells in culture. Cells of one such revertant, XP129, were previously shown to remove (6-4) photoproducts from irradiated DNA, but to have poor repair of cyclobutane pyrimidine dimers. To analyze the biochemical nature of the reversion, whole cell extracts were prepared from the SV40-immortalized fibroblast cell lines XP12RO (an XP-A cell line), the revertant XP129 (derived from XP12RO), and 1BR.3N (from a normal individual). The ability of extracts to carry out repair synthesis in UV-irradiated DNA was examined, and immunoblots were performed using antiserum that recognizes XP-A protein. XP12RO extracts exhibited a very low level of repair and no detectable XP-A protein, but repair activity could be conferred by adding purified XP-A protein to the reaction mixture. XP129 extracts have essentially normal repair synthesis consistent with the observation that most repair of UV-irradiated DNA by extracts appears to occur at (6-4) photoproducts. An XP-A polypeptide of normal size was present in XP129, but in reduced amounts. The results indicate that in XP129 a mutational event has converted the inactive XP12RO XP-A gene into a form which expresses an active XP-A protein.     

Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.     

Cell cycle localization, dimerization, and binding domain architecture of the telomere protein cPot1

Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.     

Repair of 4-nitroquinoline-1-oxide-induced DNA damage in normal human cells and cells from classical and variant xeroderma pigmentosum

The effect of 4-nitroquinoline-1-oxide (4NQO) upon 3 fibroblast cell lines derived from normal and xeroderma pigmentosum subjects have been compared. Excision-deficient XP cells (XP2BI), complementation group G, are nearly 200-fold more sensitive than normal cells to the lethal effect of 4NQO while XP variants (XP7TA), are 2-fold more sensitive. This cytotoxicity correlates with the levels of unscheduled DNA synthesis performed by the 3 cell lines. 4NQO causes a dose-related inhibition of DNA replication in all cell lines. However, newly replicated DNA synthesised immediately after treatment of cells with 4NQO is slightly smaller in XP7TA variant cells than in normal cells receiving the same dose of 4NQO, but DNA fragments in excision-deficient XP2BI are 50% smaller. It is likely that replicon elongation and joining together of newly replicated DNA fragments is dependent upon the excision of certain 4NQO-induced lesions, possibly normally repaired by a 'short-patch' repair process defective in XP2BI.     

Mammalian mitochondrial endonuclease activities specific for ultraviolet-irradiated DNA.

Mitochondrial forms of uracil DNA glycosylase and UV endonuclease have been purified and characterized from the mouse plasmacytoma cell line, MPC-11. As in other cell types, the mitochondrial uracil DNA glycosylase has properties very similar to those of the nuclear enzyme, although in this case the mitochondrial activity was also distinguishable by extreme sensitivity to dilution. Three mitochondrial UV endonuclease activities are also similar to nuclear enzymes; however, the relative amounts of these enzyme activities in the mitochondria is significantly different from that in the nucleus. In particular, mitochondria contain a much higher proportion of an activity analogous to UV endonuclease III. Nuclear UV endonuclease III activity is absent from XP group D fibroblasts and XP group D lymphoblasts have reduced, but detectable levels of the mitochondrial form of this enzyme. This residual activity differs in its properties from the normal mitochondrial form of UV endonuclease III, however. The presence of these enzyme activities which function in base excision repair suggests that such DNA repair occurs in mitochondria. Alternatively, these enzymes might act to mark damaged mitochondrial genomes for subsequent degradation.     

The DNA-binding domain of human c-Abl tyrosine kinase promotes the interaction of a HMG chromosomal protein with DNA.

The biological activity of the c-Abl protein is linked to its tyrosine kinase and DNA-binding activities. The protein, which plays a major role in the cell cycle response to DNA damage, interacts preferentially with sequences containing an AAC motif and exhibits a higher affinity for bent or bendable DNA, as is the case with high mobility group (HMG) proteins. We have compared the DNA-binding characteristics of the DNA-binding domain of human c-Abl and the HMG-D protein from Drosophila melanogaster. c-Abl binds tightly to circular DNA molecules and potentiates the interaction of DNA with HMG-D. In addition, we used a series of DNA molecules containing modified bases to determine how the exocyclic groups of DNA influence the binding of the two proteins. Interfering with the 2-amino group of purines affects the binding of the two proteins similarly. Adding a 2-amino group to adenines restricts the access of the proteins to the minor groove, whereas deleting this bulky substituent from guanines facilitates the protein-DNA interaction. In contrast, c-Abl and HMG-D respond very differently to deletion or addition of the 5-methyl group of pyrimidine bases in the major groove. Adding a methyl group to cytosines favours the binding of c-Abl to DNA but inhibits the binding of HMG-D. Conversely, deleting the methyl group from thymines promotes the interaction of the DNA with HMG-D but diminishes its interaction with c-Abl. The enhanced binding of c-Abl to DNA containing 5-methylcytosine residues may result from an increased propensity of the double helix to denature locally coupled with a protein-induced reduction in the base stacking interaction. The results show that c-Abl has unique DNA-binding properties, quite different from those of HMG-D, and suggest an additional role for the protein kinase.     

The relationship between group C adenovirus tumor antigen and the adenovirus single-strand DNA-binding protein

The group C adenoviruses code for a single-strand specific DNA-binding protein of molecular weight 72,000 daltons which is synthesized at early times after productive viral infection. Experiments were designed to determine whether this single-strand specific DNA-binding protein was expressed in adenovirus tumors and transformed cells.Two independently derived preparations of antisera from hamsters bearing group C adenovirus tumors were tested for antibody against the single-strand DNA-binding proteins. One antiserum contained antibodies that reacted with these DNA-binding proteins, while the second antiserum did not contain detectable levels of antibody. Five adenovirus type 2 transformed rat cell lines were tested for the presence of the single-strand specific DNA-binding proteins. Two of the five transformed cells expressed detectable levels of this protein. These results indicate that the group C adenovirus single-strand specific DNA-binding proteins are expressed in some, but not all, adenovirus tumors and transformed cell lines.Those transformed cell lines (type 2) containing a portion of the adenovirus genome designated by the Eco R-I-B restriction enzyme fragment express the single-strand specific DNA-binding proteins. Those cell lines missing this Eco R-I-B fragment do not contain this viral protein. Other experiments have located the structural gene of the single-strand specific DNA-binding protein in the Eco R-I-B DNA fragment, indicating that when this gene is present in a transformed cell, it is expressed.     

An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8.

An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences.     

Xeroderma pigmentosum fibroblasts of the D group lack an apurinic DNA endonuclease species with a low apparent Km.

Apurinic DNA endonuclease activity from cultured human fibroblasts was resolved into two species by phosphocellulose chromatography. The species had sedimentation coefficients of 3.3 S and 2.8 S and apparent Km's for apurinic sites of 5 and 44 nM, respectively. The low Km species was absent from extracts of cell lines XP5BE, XP6BE and XP7BE of xeroderma pigmentosum complementation group D.     

p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin.     

DNA binding by polycomb-group proteins: searching for the link to CpG islands

Polycomb group proteins predominantly exist in polycomb repressive complexes (PRCs) that cooperate to maintain the repressed state of thousands of cell-type-specific genes. Targeting PRCs to the correct sites in chromatin is essential for their function. However, the mechanisms by which PRCs are recruited to their target genes in mammals are multifactorial and complex. Here we review DNA binding by polycomb group proteins. There is strong evidence that the DNA-binding subunits of PRCs and their DNA-binding activities are required for chromatin binding and CpG targeting in cells. In vitro, CpG-specific binding was observed for truncated proteins externally to the context of their PRCs. Yet, the mere DNA sequence cannot fully explain the subset of CpG islands that are targeted by PRCs in any given cell type. At this time we find very little structural and biophysical evidence to support a model where sequence-specific DNA-binding activity is required or sufficient for the targeting of CpG-dinucleotide sequences by polycomb group proteins while they are within the context of their respective PRCs, either PRC1 or PRC2. We discuss the current knowledge and open questions on how the DNA-binding activities of polycomb group proteins facilitate the targeting of PRCs to chromatin.     

The expression of the Escherichia coli uvrA gene in human cells

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt+ colonies were selected, and cell lines established. Several lines were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95,000-dalton UvrA-related protein. The expression of uvrA was found to be 50-100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the "mammalian" UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA+ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA+ cell lines were examined for their UV-resistant phenotype and not unexpectedly were found not to be reverted to a state of repair proficiency.     

Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.     

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts.

Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal.