Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.     

Binding of yeast a1 and alpha 2 as a heterodimer to the operator DNA of a haploid-specific gene

The mating-type locus (MAT) encodes several DNA-binding proteins, which determine the three cell types of Saccharomyces cerevisiae: the a and alpha haploid cell types, and the a/alpha diploid cell type. One of the products of MAT, alpha 2, functions in two cell types. In alpha cells, alpha 2 represses the a-specific genes by binding to the operator as a dimer. In a/alpha diploid cells, alpha 2 acts with a1, a product of the other MAT allele, to repress a different set of genes, the haploid-specific genes. Until now, the nature of the interaction between a1 and alpha 2 was not known, although it had been suggested that alpha 2 may form a heterodimer with a1. I show, by using proteins synthesized in vitro, that a1 and alpha 2 bind the operator of a haploid-specific gene as a heterodimer. The ability of alpha 2 to form both homodimers and heterodimers with a1, each with a different DNA-binding specificity, explains the dual regulatory functions of alpha 2. This is the first example of regulation by heterodimerization among homeobox-containing proteins, a class that includes proteins responsible for the specification of segment identity in Drosophila, mammals and other eukaryotes.     

Local destabilisation of a DNA double helix by a T--T wobble pair.

Nuclear magnetic resonance is a technique which permits direct observation of the Waton--Click hydrogen-bonded ring imino protons (guanine N1H and thymine N3H). As the formation and disruption of hydrogen bonds of double-helical RNA and DNA structures are key events during various biological processes, NMR thus provides a useful tool for studying the fluctuational mobility of the individual base pairs. Indeed, several NMR studies of oligo- and polynucleotides have been carried out to probe the structure and dynamics of nucleic acids in solution (for a review see ref. 1). The present study constitutes the first part of our attempt to assess the influence of non-complementary base pairs on the stability of nucleic acid double helices. We report the spectral assignment and temperature-dependent NMR profiles of the hydrogen-bonded imino protons of the two DNA fragments shown in Fig. 1. The assignment is based solely on experimental grounds using the principle of chemical modification. It will be demonstrated that the introduction of a non-complementary (wobble) base pair in a DNA duplex introduces an extra melting site in addition to the sequential melting which starts with the terminal base pairs in the double helix structure.     

p53 and DNA polymerase alpha compete for binding to SV40 T antigen

The large T antigen (T) of simian virus 40 is a multifunctional protein required for both viral DNA replication and cellular transformation. T antigen forms specific protein complexes with the host protein p53 in both virus-infected and transformed cells. p53 has recently been shown to be an oncogene, but its normal function is not clear. We previously established a radioimmunoassay to study the newly described complex between T antigen and DNA polymerase alpha, and have noted a similarity between the antigenic changes induced in T by the binding of both p53 and polymerase. We now extend this analysis to a larger collection of anti-T antibodies and formally establish that p53 and DNA polymerase alpha can compete for binding to the SV40 T antigen. At a critical concentration of the three components it is possible to detect a trimeric complex of T, p53 and DNA polymerase alpha. Our observations have important implications for the control by these nuclear oncogenes of viral and cellular DNA synthesis and viral host range in both normal and transformed cells. We present a model for the action of p53 in growth control.     

Was the loss of the D helix in alpha globin a functionally neutral mutation?

Proteins in the globin family are found in a variety of species from bacteria to man. From the many globin sequences known, evolutionary trees have been constructed showing that alpha and beta globins diverged from a common ancestor between 425 and 500 million years ago, after vertebrate species had appeared and roughly when sharks and bony vertebrates diverged. The alpha and beta globins assemble to form tetrameric haemoglobin, alpha 2 beta 2, which can switch between quaternary states having high and low oxygen affinity. This allows the protein to bind oxygen cooperatively and therefore efficiently transport oxygen from the lungs to respiring tissues. The alpha and beta globins have closely related tertiary structures, being alpha-helical proteins with similar haem-binding sites. Most globins consist of eight helices, designated A to H from the N terminus, connected by short nonhelical segments, but all known vertebrate alpha globins lack a D helix. Because the loss of this helix by alpha globin occurred shortly before tetrameric haemoglobin appeared, it might be a functionally important mutation required for a tetramer assembly or allostery. We have now tested this idea by engineering human haemoglobins containing beta subunits without a D helix and alpha subunits with a D helix. Both of these mutations have little effect on the oxygen-binding properties of the molecule. Thus it is possible that deletion of the D helix in the alpha subunit was caused by a neutral mutation.     

Activation in vitro of sequence-specific DNA binding by a human regulatory factor

The human heat-shock factor (HSF) regulates heat-shock genes in response to elevated temperature. When human cells are heated to 43 degrees C, HSF is modified post-translationally from a form that does not bind DNA to a form that binds to a specific sequence (the heat-shock element, HSE) found upstream of heat-shock genes. To investigate the transduction of the heat signal to HSF, and more generally, how mammalian cells respond at the molecular level to environmental stimuli, we have developed a cell-free system that exhibits heat-induced activation of human HSF in vitro. Comparison of HSF activation in vitro and in intact cells suggests that the response of human cells to heat shock involves at least two steps. First, an ATP-independent, heat-induced alteration of HSF allows it to bind the HSE; the temperature at which activation occurs in vitro implies that a human factor directly senses temperature. Second, HSF is phosphorylated. It is possible that similar multi-step activation mechanisms play a role in the response of eukaryotic cells to a variety of environmental stimuli, and that these mechanisms evolved to increase the range and flexibility of the response.     

RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO

The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.     

Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel

Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.     

用AFM观察小白鼠心肌核DNA片段的基因体外转录和垃圾DNA的相互作用

用AFM直接观察、体外转录等实验技术组合,发现小白鼠(Balb/C)心肌体外转录状态的核DNA片段上的各种基因,处于垃圾DNA的特定的“转录平台”上。“转录平台”上的各种核活性基因的两端的调控序列,分别与特定开关蛋白质复合体结合(即可解离的开关蛋白质),中间的编码序列分别以非共价键特异结合可完全解离的转录活性因子等多种蛋白质;这些与核基因转录相关的蛋白质均由垃圾DNA的专一性蛋白质通路分别进行特异性正负反馈调控。     

Myb DNA binding inhibited by phosphorylation at a site deleted during oncogenic activation

The c-Myb nuclear oncoprotein is phosphorylated in vitro and in vivo at an N-terminal site near its DNA-binding domain by casein kinase II (CK-II) or a CK-II-like activity. This in vitro phosphorylation reversibly inhibits the sequence-specific binding of c-Myb to DNA. The site of this phosphorylation is deleted in nearly all oncogenically activated Myb proteins, resulting in DNA-binding that is independent of CK-II. Because CK-II activity is modulated by growth factors, loss of the site could uncouple c-Myb from its normal physiological regulator.     

Primary structure of helix C to helix G of a new retinal protein in H.sp.xz515

The gene fragment encoding the retinal protein from helix C to helix G in a new strain of halobacteria, H.sp.xz515 has been amplified by PCR method. The nucleotide sequence of this fragment has been determined. The deduced amino acid sequence has been compared with halobium br and other two br-like proteins, ar-1 and ar-2. Results show that those amino acid residues in br, essential for proton pumping and binding to retinal, are conserved. The residue M145 in br may be important for isomerization reaction of retinal.