Major DNA-binding Protein Research Articles

Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods

The pharmacological suppression of the DNA damage response and DNA repair can increase the therapeutic indices of conventional chemotherapeutics. Replication Protein A (RPA), the major single-stranded DNA binding protein in eukaryotes, is required for DNA replication, DNA repair, DNA recombination, and DNA damage response signaling. Through the use of high-throughput screening of 1500 compounds, we have identified a small molecule inhibitor, 15-carboxy-13-isopropylatis-13-ene-17,18-dioic acid (NSC15520), that inhibited both the binding of Rad9–GST and p53–GST fusion proteins to the RPA N-terminal DNA binding domain (DBD), interactions that are essential for robust DNA damage signaling. NSC15520 competitively inhibited the binding of p53-GST peptide with an IC 50 of 10 μM. NSC15520 also inhibited helix destabilization of a duplex DNA (dsDNA) oligonucleotide, an activity dependent on the N-terminal domain of RPA70. NSC15520 did not inhibit RPA from binding single-stranded oligonucleotides, suggesting that the action of this inhibitor is specific for the N-terminal DBD of RPA, and does not bind to DBDs essential for single-strand DNA binding. Computer modeling implicates direct competition between NSC15520 and Rad9 for the same binding surface on RPA. Inhibitors of protein–protein interactions within the N-terminus of RPA are predicted to act synergistically with DNA damaging agents and inhibitors of DNA repair. Novel compounds such as NSC15520 have the potential to serve as chemosensitizing agents.

Abstract 1009: Critical role of Pin1 for the regulation of TRF1 stability and ubiquitination

Abstract Pin1 is a peptidyl-prolyl cis-trans isomerase (PPIase) that isomerizes only phosphorylated Ser/Thr-Pro peptide bonds. These Pin1-catalyzed conformational changes after phosphorylation can have profound effects on many key proteins in diverse cellular processes. Importantly, Pin1 is tightly regulated normally by multiple mechanisms in response to upstream signals and it often acts on multiple targets to help drive certain cellular processes in one direction under a given condition synergistically. Moreover, Pin1 is highly overexpressed in many human cancers including breast cancer and is important for the activation of multiple oncogenic pathways, indicating that Pin1 plays a key role in the pathogenesis of cancer. TRF1 is a major telomeric DNA-binding protein that negatively controls telomere length. Moreover, TRF1 levels increase at entry into mitosis, and then decreased as cells exit from mitosis, and that TRF1 is important for mitotic regulation. Furthermore, TRF1 is tightly regulated by the Fbx4-mediated ubiquitination pathway and its stability can have a dramatic impact on telomere length and cell growth. In addition, TRF1 is often reduced and correlated with poor clinical outcome in many tumors. However, it is not known whether the Fbx4-mediated proteolytic pathway is regulated. Here, we report that TRF1 stability is regulated by Pin1-mediated prolyl isomerization. Pin1 interacts with the conserved Thr149-Pro motif in TRF1 in a phosphorylation-dependent manner. Inhibition of endogenous Pin1 function via multiple approaches renders TRF1 fully resistant to protein ubiquitination and degradation by Fbx4. We have also examined stability and ubiquitination of the TRF1 point mutant (TRF1 T149A) that fails to bind Pin1. This TRF1 mutant is completely stable not only in Pin1+/+ cells, but also in Pin1-/- cells. Moreover, no ubiquitinated TRF1 mutant has been detected in the presence or absence of Pin1, indicating that the Pin1-binding site in TRF1 is essential for Pin1 to promote ubiquitin-mediated degradation of TRF1. In addition, amounts of TRF1 protein are increased in tissues examined from Pin1-deficient mice, in comparison with those from wild-type mice, indicating that TRF1 level is upregulated in Pin1 knockout mice. Furthermore, TRF1 expression is decreased and correlated with Pin1 overexpression in human breast cancer tissues, suggesting that a close relationship between Pin1 and TRF1 levels under both physiological and pathological conditions. Thus, Pin1 is a novel regulator of TRF1 and its overexpression might contribute to the downregulation of TRF1 in tumors such as breast cancer. Given that the Pin1 targeting pThr149-Pro motif is located in the middle of the TRF1 homology domain that also interacts with Fbx4, Pin1-catalyzed conformational changes might regulate the interaction between TRF1 and Fbx4 to promote Fbx4-mediated degradation of TRF1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1009.

Abstract 4566: Triplex DNA-binding proteins in resected normal and tumor tissues from colorectal cancer patients: Biological and clinical relevance

Abstract This study is to determine if triplex DNA-binding proteins in human cells play an important role in processes that lead to cancer such as genetic instability or growth deregulation. Cytoplasmic and nuclear extracts from 60 colorectal cancer patients’ tumor (T) and normal (N) tissues were isolated and examined by gel shifts (EMSA) for triplex DNA-binding proteins. Total tumor extracts showed significantly higher triplex DNA-binding activities of the major H3 complex than normal tissue extracts (Wilcoxon test). Neither T-staging nor M-staging at diagnosis was associated with any of the factors, but N-Stage was significantly associated with the ratio of tumor/normal (T/N) for the total of cytoplasmic and nuclear extracts. This means all patients without lymph node affectation had decreased binding ratios (T/N) in both cytoplasmic and nuclear extracts. Kaplan-Meier survival analysis using a cut-off of 1.5 (rounded-up median) for the nuclear binding activity ratio (T/N) showed significantly poorer survival for those patients whose nuclear binding was greater than 1.5. This suggests that although the major triplex DNA-binding protein is present in normal tissue extracts, it is more abundant in tumor extracts, and that a specific abundance of the major triplex-binding protein (H3) in the tumor nucleus is associated with poorer patient survival. Biotinylated triplex DNA-protein complexes were isolated from the RKO colorectal cancer cell line with streptavidin-agarose and analyzed by nano-HPLC ESI-MS-MS. We identified 100 and 60 kDa proteins from nuclear extracts as PSF (polypyrimidine tract binding-associated splicing factor) and p54nrb (NonO), known to function as RNA polymerase II-associated splicing factors, bind as heterodimers, and implicated in the regulation of expression of the myc family of oncoproteins and COX2. We also identified a 75 kDa protein from cytoplasmic extracts as U2AF65 (U2 small nuclear RNA auxiliary factor 2 isoform b), also a splicing factor that can directly interact with p54 nrb and shuttle continuously between the nucleus and cytoplasm. To examine differences between tumor and normal tissues in multiple signaling proteins and proteins known to be relevant in colorectal cancer, reverse phase protein array (RPPA), Pearson correlation and cluster analysis of tumor nuclear lysates ranked from lowest to highest H3 EMSA values shows a highly significant correlation between decreased β-catenin expression in tissues that have the lowest H3 EMSA values and increased β-catenin expression in tissues that have the highest H3 EMSA values. We found this pattern in all tissue lysate types: normal-cytoplasm, normal-nucleus, tumor-cytoplasm, and tumor-nucleus. We believe these splicing factors are overexpressed in tumor tissues and could cause deregulated growth of cells by influencing other proteins associated with tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4566.

Morphology and protein composition of the mitochondrial nucleoids in yeast cells lacking Abf2p, a high mobility group protein

To elucidate the role of Abf2p, a major mitochondrial DNA-binding protein in the yeast Saccharomyces cerevisiae, we examined the morphology of the mitochondrial nucleoids (mt-nucleoids) in an ABF2-deficient mutant (Δabf2) in vivo and in vitro by 4',6-diamidino-2-phenylindole (DAPI) staining. The mt-nucleoids appeared as diffuse structures with irregular-size in Δabf2 cells that were grown to log phase in YPG medium containing glycerol, in contrast to the strings-of-beads appearance of mt-nucleoids in wild-type cells. In addition, DAPI-fluorescence intensity of the mt-nucleoids transmitted to the bud was significantly lower in Δabf2 cells than in wild-type cells at log phase. However, the lack of Abf2p did not affect the morphology or segregation of mitochondria. The protein composition of the mt-nucleoids isolated from Δabf2 cells grown to stationary phase in YPG medium was very similar to that of the mt-nucleoids isolated from wild-type cells cultured under the same conditions, except for the lack of Abf2p. These results together suggested that in log-phase cells, the lack of Abf2p influences not only the morphology of mt-nucleoids but also their transmission into the bud. On the other hand, our result suggested that in stationary-phase cells, the lack of Abf2p does not significantly alter the protein composition of the mt-nucleoids.

How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA

The POT1-TPP1 heterodimer, the major telomere-specific single-stranded DNA-binding protein in mammalian cells, protects chromosome ends and contributes to the regulation of telomerase. The recent discovery of telomeric RNA raises the question of how POT1 faithfully binds telomeric ssDNA and avoids illicit RNA binding that could result in its depletion from telomeres. Here we show through binding studies that a single deoxythymidine in a telomeric repeat dictates the DNA versus RNA discrimination by human POT1 and mouse POT1A. We solve the crystal structure of hPOT1 bound to DNA with a ribouridine in lieu of the critical deoxythymidine and show that this substitution results in burying the 2(')-hydroxyl group in a hydrophobic region (Phe62) of POT1 in addition to eliminating favorable hydrogen-bonding interactions at the POT1-nucleic acid interface. At amino acid 62, Phe discriminates against RNA binding and Tyr allows RNA binding. We further show that TPP1 greatly augments POT1's discrimination against RNA.

Regulatory Functions of the N-terminal Domain of the 70-kDa Subunit of Replication Protein A (RPA)

Replication protein A (RPA) is the major single-stranded DNA-binding protein in eukaryotes. RPA is composed of three subunits of 70, 32, and 14 kDa. The N-terminal domain of the 70-kDa subunit (RPA70) has weak DNA binding activity, interacts with proteins, and is involved in cellular DNA damage response. To define the mechanism by which this domain regulates RPA function, we analyzed the function of RPA forms containing a deletion of the N terminus of RPA70 and mutations in the phosphorylation domain of RPA (N-terminal 40 amino acids of the 32-kDa subunit). Although each individual mutation has only modest effects on RPA activity, a form combining both phosphorylation mimetic mutations and a deletion of the N-terminal domain of RPA70 was found to have dramatically altered activity. This combined mutant was defective in binding to short single-stranded DNA oligonucleotides and had altered interactions with proteins that bind to the DNA-binding core of RPA70. These results indicate that in the absence of the N-terminal domain of RPA70, a negatively charged phosphorylation domain disrupts the activity of the core DNA-binding domain of RPA. We conclude that the N-terminal domain of RPA70 functions by interacting with the phosphorylation domain of the 32-kDa subunit and blocking undesirable interactions with the core DNA-binding domain of RPA. These studies indicate that RPA conformation is important for regulating RPA-DNA and RPA-protein interactions.

Purification of an Abf2p-like protein from mitochondrial nucleoids of yeast Pichia jadinii and its role in the packaging of mitochondrial DNA

A 26-kDa protein with highly basic pI was purified from the mitochondrial (mt-) nucleoids of the yeast Pichia jadinii by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 26-kDa protein has the ability to introduce a supercoil into circular plasmid DNA in the presence of topoisomerase I and to package mtDNA into nucleoid-like aggregates. The mt-nucleoids isolated from P. jadinii cells were disassembled in the presence of 2 M NaCl and reassembled into nucleoid-like aggregates by the removal of the salts. During the course of the reassembly of the mt-nucleoids, three specific proteins of 20 kDa, 26 kDa and 56 kDa predominantly precipitated after the centrifugation of the reassembled mt-nucleoids. These results suggest that the 26-kDa protein of P. jadinii has a similar function in the packaging of mtDNA to Abf2p, a major mitochondrial DNA-binding protein in Saccharomyces cerevisiae.

Phosphorylation of replication protein A by S-phase checkpoint kinases

The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Here we show that human RPA70 and RPA32 can be phosphorylated by purified S-phase checkpoint kinases, ATR and Chk1. While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain. Chk1 phosphorylated RPA70 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation, suggesting that Chk1 and ssDNA compete for RPA's major ssDNA binding domain. ssDNA stimulates RPA32 phosphorylation by ATR in a length dependent manner. Furthermore, 3′-, but not 5′-, recessed single strand/double strand DNA junctions produce an even stronger stimulatory effect on RPA32 phosphorylation by ATR. This stimulation occurs for both RNA and DNA recessed ends. RPA's DNA binding polarity and its interaction to 3′-primer-template junctions contribute to efficient RPA32 phosphorylation. Progression of DNA polymerase is able to block the accessibility of the 3′-recessed ends and prevent the stimulatory effects of primer-template junctions on RPA phosphorylation by ATR. We propose models for the role of RPA phosphorylation by Chk1 in S-phase checkpoint pathways, and the possible regulation of ATR activity by different nucleic acid structures.

Evidence of Meiotic Crossover Control in Saccharomyces cerevisiae Through Mec1-Mediated Phosphorylation of Replication Protein A

Replication protein A (RPA) is the major single-stranded DNA-binding protein in eukaryotes, essential for DNA replication, repair, and recombination. During mitosis and meiosis in budding yeast, RPA becomes phosphorylated in reactions that require the Mec1 protein kinase, a central checkpoint regulator and homolog of human ATR. Through mass spectrometry and site-directed mutagenesis, we have now identified a single serine residue in the middle subunit of the RPA heterotrimer that is targeted for phosphorylation by Mec1 both in vivo and in vitro. Cells containing a phosphomimetic version of RPA generated by mutation of this serine to aspartate exhibit a significant alteration in the pattern of meiotic crossovers for specific genetic intervals. These results suggest a new function of Mec1 that operates through RPA to locally control reciprocal recombination.

The Crystal Structure of the Herpes Simplex Virus 1 ssDNA-binding Protein Suggests the Structural Basis for Flexible, Cooperative Single-stranded DNA Binding

All organisms including animal viruses use specific proteins to bind single-stranded DNA rapidly in a non-sequence-specific, flexible, and cooperative manner during the DNA replication process. The crystal structure of a 60-residue C-terminal deletion construct of ICP8, the major single-stranded DNA-binding protein from herpes simplex virus-1, was determined at 3.0 A resolution. The structure reveals a novel fold, consisting of a large N-terminal domain (residues 9-1038) and a small C-terminal domain (residues 1049-1129). On the basis of the structure and the nearest neighbor interactions in the crystal, we have presented a model describing the site of single-stranded DNA binding and explaining the basis for cooperative binding. This model agrees with the beaded morphology observed in electron micrographs.

Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

Peptide mimics of a major lupus epitope of SmB/B'.

One of the most distinguishing features of systemic lupus erythematosus is the presence of high concentrations of autoantibodies that recognize a limited number of self-antigens. Even though many lupus autoantigens have been identified, the inciting triggers of these abnormal immune responses are not fully understood. One mechanism that could generate these autoantibodies is a normal immune response toward a foreign epitope that mimics a common antigenic target of an autoantigen. Antibody generated toward the foreign epitope could also bind the autoantigen. This "cross-reactivity" would result in the presentation of the autoantigen to the immune system. Under autoimmune-prone conditions, tolerance toward the native protein is broken and an autoimmune response is initiated. Previously, it was suggested that Epstein-Barr virus might use such a mechanism to initiate an autoimmune response. Cross-reactive epitopes may have a similar amino acid sequence or a similar tertiary structure that is independent of amino acid sequence. A major, and likely initial, target of the lupus anti-SmB' response is a repeated, proline-rich sequence, PPPGMRPP. To identify potential cross-reactive targets, we used affinity-purified autoantibodies specific for PPPGMRPP to screen a random heptapeptide phage display library. Eighty-five clones were isolated and sequenced with eleven distinct sequence motifs being identified. Two of these motifs were homologous to the SmB' epitope, while the other nine were not. Interestingly, one of the peptide motifs that mimicked the SmB' epitope is identical to a peptide sequence found in the Epstein-Barr virus major DNA binding protein.

Organization, Developmental Dynamics, and Evolution of Plastid Nucleoids

The plastid is a semiautonomous organelle essential in photosynthesis and other metabolic activities of plants and algae. Plastid DNA is organized into the nucleoid with various proteins and RNA, and the nucleoid is subject to dynamic changes during the development of plant cells. Characterization of the major DNA-binding proteins of nucleoids revealed essential differences in the two lineages of photosynthetic eukaryotes, namely nucleoids of green plants contain sulfite reductase as a major DNA-binding protein that represses the genomic activity, whereas the prokaryotic DNA-binding protein HU is abundant in plastid nucleoids of the rhodophyte lineage. In addition, current knowledge on DNA-binding proteins, as well as the replication and transcription systems of plastids, is reviewed from comparative and evolutionary points of view. A revised hypothesis on the discontinuous evolution of plastid genomic machinery is presented: despite the cyanobacterial origin of plastids, the genomic machinery of the plastid genome is fundamentally different from its counterpart in cyanobacteria.

Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins.

Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids. However, the protein profile of the mt-nucleoids of K. lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae. SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S. cerevisiae on a gel, detected only a 17-kDa protein in the K. lactis mt-nucleoid proteins. The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S. cerevisiae Abf2p, and it packed K. lactis mtDNA into nucleoid-like particles in vitro. These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K. lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.

Memory Cytotoxic T Cell Responses to Viral Tegument and Regulatory Proteins Encoded by Open Reading Frames 4, 10, 29, and 62 of Varicella-Zoster Virus

Cytotoxic T cell recognition of tegument and regulatory proteins encoded by open reading frames (ORFs) 4, 10, 29, and 62 of varicella-zoster virus (VZV) was evaluated using limiting dilution conditions to estimate the precursor frequencies of memory T cells specific for these proteins in immune subjects. Responder cell frequencies for ORFs 4, 10, and 62 gene products, which are virion tegument components and function as immediate early viral transactivating proteins, were equivalent. CTLp recognition of VZV proteins made in latently infected cells, which include ORF4 and ORF62 proteins, was not maintained preferentially when compared to ORF10 protein, which has not been shown to be expressed during latency. T cell recognition of ORF29 protein, the major DNA binding protein, which is expressed during replication but not incorporated into the virion tegument, was less common than responses to ORFs 4, 10, and 62 gene products. Older individuals had diminished numbers of memory CTLp that lysed autologous targets expressing IE62 protein; these responses were increased after immunization with live attenuated varicella vaccine to the range observed in younger adults. Adaptive immunity to VZV is characterized by a broad repertoire of memory CTL responses to proteins that comprise the virion tegument and regulate viral gene expression in infected cells.

Characterization of varicella-zoster virus gene 21 and 29 proteins in infected cells.

Varicella-zoster virus (VZV) transcription is limited in latently infected human ganglia. Note that much of the transcriptional capacity of the virus genome has not been analyzed in detail; to date, only VZV genes mapping to open reading frames (ORFs) 4, 21, 29, 62, and 63 have been detected. ORF 62 encodes the major immediate-early virus transcription transactivator IE62, ORF 29 encodes the major virus DNA binding protein, and ORF 21 encodes a protein associated with the developing virus nucleocapsid. We analyzed the cellular location of proteins encoded by ORF 21 (21p) and ORF 29 (29p), their phosphorylation state during productive infection, and their ability form a protein-protein complex. The locations of both 21p and 29p within infected cells mimic those of their herpes simplex virus type 1 (HSV-1) homologues (UL37 and ICP8); however, unlike these homologues, 21p is not phosphorylated and neither 21p nor 29p exhibits a protein-protein interaction. Transient transfection assays to determine the effect of 21p and 29p on transcription from VZV gene 20, 21, 28, and 29 promoters revealed no significant activation of transcription by 21p or 29p from any of the VZV gene promoters tested, and 21p did not significantly modulate the ability of IE62 to activate gene transcription. A modest increase in IE62-induced activation of gene 28 and 29 promoters was seen in the presence of 29p; however, IE62-induced activation of gene 28 and 29 promoters was reduced in the presence of 21p. A Saccharomyces cerevisiae two-hybrid analysis of 21p indicated that the protein can activate transcription when tethered within a responsive promoter. Together, the data reveal that while VZV gene 21 and HSV-1 UL37 share homology at the nucleic acid level, these proteins differ functionally.

Identification of the YMN-1 antigen protein and biochemical analyses of protein components in the mitochondrial nucleoid fraction of the yeast Saccharomyces cerevisiae.

We analyzed the protein components contained in the mitochondrial nucleoid (mt-nucleoid) fraction of the yeast Saccharomyces cerevisiae. Immunoblotting with anti-Abf2p antibody demonstrated the association of Abf2p, a major mitochondrial DNA-binding protein, with the mt-nucleoids. In contrast, porin and cytochrome c oxidase subunit III (CoxIIIp) were not detected by immunoblotting in the mt-nucleoid fraction. The YMN-1 monoclonal antibody recognized a 48 kDa protein of the mt-nucleoid fraction. The N-terminal amino acid sequence of the protein and immunological evidence showed that the YMN-1 monoclonal antibody recognizes dihydrolipoyl transsuccinylase (KE2), which is one of the constituents of the alpha-ketoglutarate dehydrogenase complex (KGDC). alpha-Ketoglutarate dehydrogenase (KE1) and dihydrolipoyl dehydrogenase (E3), which are other subunits of KGDC, were also detected in the mt-nucleoid fraction. An enzyme assay of the mt-nucleoid fraction showed that cytochrome c oxidase and fumarase activity were barely detected in the fraction, but the specific activity of KGDC in the mt-nucleoid fraction was relatively high and was approximately 60% of the specific activity in the mitochondrial fraction. Three components of KGDC were detected in the DNA-binding protein fractions after DNA-cellulose column chromatography of mt-nucleoid proteins. These results suggested that a part of KGDC in the mitochondrial matrix is associated with mt-nucleoids in vivo.

Conformational Change in the Herpes Simplex Single-Strand Binding Protein Induced by DNA

Protease digestion of the herpes simplex virus type 1 major single-strand DNA binding protein ICP8 showed that the cleavage patterns observed in the presence and absence of single-stranded DNA oligonucleotides are substantially different with protection of cleavage sites between amino acids 293 and 806 observed in the presence of oligonucleotide. Experiments using ICP8 modified with fluorescein-5-maleimide (FM) showed that the fluorescence signal exhibited increased susceptibility to antibody quenching and a significant decrease in polarization of the FM fluorescence was observed in the presence compared to the absence of oligonucleotide. Taken together, these results indicate that ICP8 undergoes a conformational change upon binding to single-stranded DNA.

Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase.

Antigens of pathogenic microbes that mimic autoantigens are thought to be responsible for the activation of autoreactive T cells. Viral infections have been associated with the development of the neuroendocrine autoimmune diseases type 1 diabetes and stiff-man syndrome, but the mechanism is unknown. These diseases share glutamic acid decarboxylase (GAD65) as a major autoantigen. We screened synthetic peptide libraries dedicated to bind to HLA-DR3, which predisposes to both diseases, using clonal CD4(+) T cells reactive to GAD65 isolated from a prediabetic stiff-man syndrome patient. Here we show that these GAD65-specific T cells crossreact with a peptide of the human cytomegalovirus (hCMV) major DNA-binding protein. This peptide was identified after database searching with a recognition pattern that had been deduced from the library studies. Furthermore, we showed that hCMV-derived epitope can be naturally processed by dendritic cells and recognized by GAD65 reactive T cells. Thus, hCMV may be involved in the loss of T cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity.

Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA–DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.