NORMAL GENOMIC

• Raval-Fernandes S, Kickhoefer VA, Rome LH
• Cloning of a cDNA encoding a sequence-specific single-stranded-DNA-binding protein from Rattus norvegicus.
• Gene. 1999; 237: 201-7
• Display abstract

• In this paper, we report the isolation of a cDNA clone encoding a sequence-specific single-stranded-DNA-binding protein (SSDP) from rat (Rattus norvegicus). The full-length nucleotide sequence was determined and encodes a 361 amino acid protein with a predicted molecular mass of 37.7 kDa. This clone has approximately 80% homology to a previously isolated partial cDNA clone for SSDP from chicken (Gallus gallus). Northern blot analysis revealed two transcripts of 2.0 and 3.0 kb. The protein appears to be evolutionarily highly conserved with > 97% identity between chicken, rat, mouse and human. Chicken SSDP has been proposed to be involved in the transcriptional regulation of the alpha 2(I) collagen gene.

• Chernov BK, Chinenov IV, Golova IB, Gurskii IG, Leinsoo TA, Gurskii GV
• [Design, synthesis, and expression of a gene coding for a protein with two DNA-binding motifs]
• Mol Biol (Mosk). 1999; 33: 779-90

• Rochester SC, Traktman P
• Characterization of the single-stranded DNA binding protein encoded by the vaccinia virus I3 gene.
• J Virol. 1998; 72: 2917-26
• Display abstract

• The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed in Escherichia coli and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.

• Chepenik LG, Tretiakova AP, Krachmarov CP, Johnson EM, Khalili K
• The single-stranded DNA binding protein, Pur-alpha, binds HIV-1 TAR RNA and activates HIV-1 transcription.
• Gene. 1998; 210: 37-44
• Display abstract

• Previous studies indicate that the bulge and loop domains of TAR, the HIV-1 RNA regulatory element, bind viral and cellular factors that are critical for efficient transcription of the HIV-1 genome. In this report, we demonstrate that the cellular protein, Pur-alpha, a previously characterized sequence specific, single-stranded DNA binding protein, binds to HIV-1 TAR RNA in a specific manner as demonstrated by competition analysis. Pur-alpha binds to the greatest extent to wild-type TAR RNA, and it appears the primary sequence, as well as the secondary structure and its overall stability contribute to this binding. Results from gel shift analysis using mutant Pur-alpha proteins indicate that amino acids 55-85, which contain the first of three basic aromatic repeats, are important for its binding to TAR RNA. Overexpression of Pur-alpha in a glial cell line increased transcription of HIV-1 LTR by a TAR dependent mechanism. The potential contribution by Pur-alpha to HIV-1 expression in relation to basal transcription by cellular factors is discussed.

• Jurk M, Weissinger F, Lottspeich F, Schwarz U, Winnacker EL
• Characterization of the single-strand-specific BPV-1 origin binding protein, SPSF I, as the HeLa Pur alpha factor.
• Nucleic Acids Res. 1996; 24: 2799-806
• Display abstract

• SPSF I and II are two cellular proteins which bind specifically to single-stranded DNA. SPSF I and II binding sites are found in the minimal origin of replication of BPV-1 DNA and near the P2 promoter of the cellular c-myc gene. DNA-binding properties of the two proteins to single-stranded oligonucleotides of different lengths and sequences were quantified by determination of DNA-binding constants. The binding constant of SPSF proteins to the lower strand of the BPV-1 origin was determined to be 1.5 x 10(-10) M-1. Peptide sequences derived from purified SPSF I and II revealed the identity of at least one of the SPSF proteins with the so-called HeLa Pur alpha factor. The HeLa Pur alpha factor was identified previously by virtue of its capacity to bind to purine-rich strands of the PUR element found in initiation zones of DNA replication [Bergemann, A.D., Ma,Z.-W. and Johnson, E.M. (1992) Mol. Cell. Biol. 12, 5673-5682]. Expression of the Pur cDNA confirmed the identity of the Pur alpha protein with the 42 kDa SPSF I protein. Analysis of several Pur alpha cDNA clones revealed the existence of an extended 3'-untranslated region in all Pur mRNAs.

• Chang CF et al.
• Evidence that replication of human neurotropic JC virus DNA in glial cells is regulated by the sequence-specific single-stranded DNA-binding protein Pur alpha.
• J Virol. 1996; 70: 4150-6
• Display abstract

• Initiation of polyomavirus DNA replication in eukaryotic cells requires the participation of the viral early protein T antigen, cellular replication factors, and DNA polymerases. The human polyomavirus JC virus (JCV) is the etiologic agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised individuals. This virus exhibits a narrow host range and a tissue specificity that restricts its replication to glial cells of the central nervous system. Restriction of viral DNA replication due to species specificity of the DNA polymerase, coupled with glial cell-specific transcription of the viral early promoter, is thought to account for the brain-specific replication of JCV. In this report we demonstrate that overexpression of Pur alpha, a protein which binds to single-stranded DNA in a sequence-specific manner, suppresses replication of JCV DNA in glial cells. Results from footprinting studies indicate that Pur alpha and T antigen share a common binding region spanning the single-stranded ori sequence of JCV. Further, T antigen was capable of stimulating the association of Pur alpha with the ori sequence in a band shift assay. Whereas no evidence for simultaneous binding of Pur alpha and T antigen to single-stranded DNA has been observed, results from coimmunoprecipitation and Western blot (immunoblot) analyses of proteins derived from cells producing JCV T antigen indicate a molecular association of JCV T antigen and Pur alpha. The binding of Pur alpha to the single-stranded ori sequence and its association with T antigen suggest that Pur alpha interferes with the activity of T antigen and/or other regulatory proteins to exert its negative effect on JCV DNA replication. The importance of these findings in the reactivation of JCV in the latently infected individual under immunosuppressed conditions is discussed.

• Johnson EM et al.
• Association of human Pur alpha with the retinoblastoma protein, Rb, regulates binding to the single-stranded DNA Pur alpha recognition element.
• J Biol Chem. 1995; 270: 24352-60
• Display abstract

• The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Pur alpha, a sequence-specific single-stranded DNA-binding protein implicated in control of gene transcription and DNA replication. These complexes can be immunoextracted from cell lysates using monoclonal antibodies to either Pur alpha or Rb. The Pur alpha-Rb complexes contain a form of Pur alpha with extensive post-synthetic modification, as demonstrated following expression of Pur alpha cDNA fused to a 9-amino acid epitope tag. Human Pur alpha, expressed as a glutathione S-transferase fusion protein, specifically binds to the hypophosphorylated form of Rb with an affinity as high as that of SV40 large T-antigen. In the absence of DNA, glutathione S-transferase-Pur alpha binds to p56RB, an NH2-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Pur alpha recognition element disrupts these complexes. Conversely, high concentrations of p56RB prevent Pur alpha binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Pur alpha is localized to a series of modular amino acid repeats. Rb binding involves a Pur alpha region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Pur alpha to p56RB, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.

• Ito K, Sato K, Endo H
• Cloning and characterization of a single-stranded DNA binding protein that specifically recognizes deoxycytidine stretch.
• Nucleic Acids Res. 1994; 22: 53-8
• Display abstract

• We previously identified a G-rich silencer element involved in negative regulation of catalase gene expression in some hepatoma cells (Mol. Cell. Biol., (1992), 12, 2525-2533). To study a nuclear binding protein for this element, we screened cDNA libraries from a rat ascites hepatoma cell line by binding with a synthetic oligonucleotide probe and obtained several clones. One of them, designated SW, was studied in detail. A clone (SW2) of this series contained a near full length cDNA encoding a putative peptide with 463 amino acid residues. We isolated this peptide as a fusion protein. It was found that the protein strongly bound to the C-stretch of the DNA sequence in a single strand specific fashion, but absolutely did not to G-rich sequence. The protein bound weakly to the corresponding double-stranded DNA as well as to C-rich RNA sequence. This protein, though not the expected one, was found to be a novel protein whose DNA binding domain was located on the region containing at least 75 amino acid residues of the carboxyl terminus. A proline rich region was also observed in the middle part of the protein. Northern blot profiles indicated extensive and slight expression of both 2.0 kb and 2.7 kb mRNA species in some hepatoma cell lines and in the rat liver, respectively.

• Ma ZW, Bergemann AD, Johnson EM
• Conservation in human and mouse Pur alpha of a motif common to several proteins involved in initiation of DNA replication.
• Gene. 1994; 149: 311-4
• Display abstract

• Pur alpha is a sequence-specific single-stranded DNA-binding protein with affinity for an element present in several eukaryotic origins of DNA replication (ori) and gene regulatory regions. We report here the cDNA sequence for mouse pur alpha and an extraordinary degree of conservation between human and mouse Pur alpha (hPurA and mPurA, respectively). There are only two single-amino-acid (aa) changes between hPurA (322 aa) and mPurA (321 aa). One PurA region of 22 aa, termed the 'psycho' motif, possesses significant homology to a counterpart in the SV40 large T-antigen, to several other transforming proteins of DNA tumor viruses, and to certain cellular proteins in yeast and human cells that may also be involved in the initiation of DNA replication.

• Negishi Y et al.
• Identification and cDNA cloning of single-stranded DNA binding proteins that interact with the region upstream of the human c-myc gene.
• Oncogene. 1994; 9: 1133-43
• Display abstract

• We have previously reported that a c-myc protein complex binds to the region upstream of the c-myc gene, where exist an origin of cellular DNA replication (ori) and a transcriptional enhancer. Both functions require a 21 bp long sequence, while the c-myc protein complex recognizes a 7 bp consensus therein. It was recently reported that single-stranded DNA binding proteins bound specifically to sequences that play roles in DNA replication or transcription. We examined for proteins binding to the single-stranded DNAs of the 21 bp element (myc(H-P)21). In a band shift assay with HL60 cells nuclear extract, probes of either the plus strand or the minus strand gave rise to specific signals. Mutation introduced within a short consensus (A/TCTA/TA/TT) present in both strands completely abolished binding in either case. Southwestern blotting analysis showed that proteins of molecular weight 105, 80, 50, 45, 40, 39.5 and 14 kDa bound sequence-specifically to either strand and 22 kDa to minus strand to the cognate A/TCTA/TA/TT consensus. These single-stranded DNA binding proteins were named MSSP, c-myc gene single strand binding proteins. We attempted to isolate the cDNAs encoding these proteins by screening a human cDNA library with the plus single-stranded oligonucleotide as a probe. Among several positive clones, we have characterized one, termed MSSP-1. MSSP-1 produced in E. coli as a fusion protein with GST specifically interacted with single-stranded TCTTAT (plus myc(H-P)21) and ACT-ATT (in minus myc(H-P)21), the consensus of which can be referred to as A/TCTA/TA/TT. Sequence analysis of MSSP-1 cDNA revealed that two domains thereof are homologous to the RNA binding motifs common to several ribonucleoproteins. Interestingly, the MSSP-1/GST fusion protein specifically recognized myc(H-P)21 not only in single-stranded but also in double-stranded forms. Binding properties of MSSP-1 imply its functions in DNA replication. Furthermore, when the AT stretch in the SV40 ori core was substituted by TCTTAT, MSSP-1 promoted viral DNA replication depending on the consensus sequences.

• Bergemann AD, Ma ZW, Johnson EM
• Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.
• Mol Cell Biol. 1992; 12: 5673-82
• Display abstract

• The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.