Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication

Dissection and reconstitution of the adenovirus DNA replication machinery has led to the discovery of two HeLa nuclear proteins which are required in conjunction with three viral proteins. One of these, nuclear factor I (NF-I), recognizes an internal region of the origin between nucleotides 25 and 40 and by binding to one side of the helix stimulates the initiation reaction up to 30-fold. NFI-binding sites have been observed upstream of several cellular genes, such as chicken lysozyme, human IgM and human c-myc, and coincide in most cases with DNase I hypersensitive regions. Here we report the identification of a novel DNA-binding protein from HeLa nuclei, designated NF-III, that recognizes a sequence in the adenovirus origin very close to the NFI-binding site, between nucleotides 36 and 54. This sequence includes the partially conserved nucleotides TATGATAATGAG. NF-III stimulates DNA replication four- to sixfold by increasing the initiation efficiency. Potential cellular binding sites include promoter elements of the histone H2B gene, the human interferon beta gene, the human and mouse immunoglobulin VK and VH genes and the mammal/chicken/Xenopus laevis U1 and U2 small nuclear RNA genes. Furthermore, a subset of the herpes simplex virus immediate early promoter specific TAATGARAT elements is homologous with the adenovirus 2 (Ad-2) NFIII-binding site.     

Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin

DNA sequences, important for the control of Drosophila heat shock gene expression, are packaged in chromatin in a nuclease hypersensitive configuration. Recently, two protein-binding (exonuclease-resistant) sites which cover the TATA box sequence and an upstream control element were shown to occur in vivo amidst the 5' terminal hypersensitive regions of several heat shock genes. Protein-binding at the TATA box is independent of heat shock, but the binding at the upstream element is heat shock dependent, and it was proposed that a heat shock activator protein, HAP, positively regulates the genes. Here, I report the detection of HAP activity in heat shocked cell extracts by reconstituting specific binding to hsp82 gene chromatin in vitro. Inhibition of the binding by free DNA from the 5' region of heat shock genes implies a coordinate regulation of the gene family through HAP interaction with the upstream heat shock consensus sequence. Furthermore, the special ease of induction of the hsp82 gene over other heat shock genes can be explained in molecular terms by the higher affinity of HAP for the hsp82 binding site, which contains a 28 base sequence with almost perfect dyad symmetry, GAAGCCTCTAGAAG/TTTCTAGAGACTTC.     

Alteration of c-myc chromatin structure by avian leukosis virus integration

The most common sites of integration of the leukosis virus (ALV) long terminal repeat (LTR) in bursal lymphomas and derivative cell lines correspond to a region encompassed by two major hypersensitive sites in the 5' flanking region of the pre-integration, unrearranged c-myc gene. After integration of the ALV LTR, the major hypersensitive site within the avian c-myc oncogene region is within the proviral LTR, and the major hypersensitive sites normally found in uninfected cells 5' to the first c-myc coding exon are no longer detectable.     

A hypersensitive site in hsp70 chromatin requires adjacent not internal DNA sequence

Nuclease-hypersensitive sites in chromatin exist at the 5' side of many eukaryotic genes. To gain some understanding of the molecular basis of these hypersensitive sites, we have now examined the pair of sites upstream of the Drosophila hsp70 gene in a series of plasmids that contain deletions in the hypersensitive region and have been transformed into yeast cells. Hypersensitive sites 5' to a Drosophila hsp70 gene are preserved when this gene is introduced into yeast by transformation. We find that a yeast strain containing a plasmid in which the deletion extends through the first hypersensitive site still displays the normal pair of hypersensitive sites, so DNA sequences over which the first hypersensitive site is centred are not required for hypersensitivity at this position and the site can form over a foreign DNA sequence juxtaposed against this deletion end point. Deletions progressing further into the region bracketed by the pair of 5' hypersensitive sites eliminate the first hypersensitive site and alter the downstream site. We propose that the hypersensitive sites are generated through the binding of a protein that renders flanking sequences more accessible to nucleases, perhaps by preventing normal chromatin packaging.     

Repetitive shuttling of a motor protein on DNA

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.     

Role for a Drosophila Myb-containing protein complex in site-specific DNA replication

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.     

The locus of sequence-directed and protein-induced DNA bending

The bending locus of trypanosome kinetoplast DNA, identified by gel electrophoresis, has tracts of a simple repeat sequence (CA5-6 T) symmetrically distributed about it, with a repeat interval of 10 base pairs. The analogous bending induced when catabolite gene activating protein binds to its recognition sequence near the promoter of the Escherichia coli lac operon is centred on a site about 5-7 base pairs away from the centre of the protein binding site.     

用计算机对人类TSPYl基因P53结合位点的鉴定

根据p53下游基因在其调节区域（启动子或内含子）含有与P53蛋白特异性结合的一致性序列5’-RRRCWWGYYYN（0-13）RRRCWWGYYY-3’，R—G或A，W—T或A，Y—C或T，N—A，C，T，G。用计算机对人类基因组中P53结合位点进行了研究，发现Y染色体上的TSPY1基因内含子中含有这样的一致性序列5’-GGGCTAGTTTtgGAGCTAGCCT-3’，意味着TSPY1基因有可能是一个p53下游基因。     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.     

短柄五加(Acanthopanax brachypus)rbcL基因的结构分析

克隆了含完整短柄五加rbcL基因的3.2kb EcoRI片段,测定了该基因的核苷酸序列.所测核苷酸序列总长度为1924bp,其中编码区1428bp,编码475个氨基酸的蛋白质.测定的基因5’上游区共278bp,包含原核性质-35区(TTGCGC),-10区(TACAAT)及类似真核的TATA box元件(TATATA).5’前导区长194bp,其中SD序列为GGAGG,紧邻起始密码子上游.测定的3’下游区共218bp,含2个相邻的转录后可形成茎环结构的反向重复序列.短柄五加rbcL基因编码区推导的氨基酸序列与烟草、菠菜、豌豆、苜蓿、玉米、水稻、松树、地钱、衣藻和Anacystis的同源性分别为93.5％、94.11％、94.53％、94.74％、89.68％、92.21％、92.21％、92.63％、87.58％和80.84％.本文还对不同植物rbcL基因的启动区及部分5’和3’非编码区进行了比较分析.