Direct evidence for DNA bending at the lambda replication origin

Replication initiation in bacteriophage lambda appears to require wrapping of origin DNA on an approximately 50 angstrom radius in or around the complex with the initiator protein O. Since short lengths of DNA are not that flexible, it may be that runs of coherently spaced deoxyadenylate residues constitute bend sites in the ori sequence that facilitate the process. Earlier data showed that ori DNA has electrophoretic anomalies characteristic of bend sites and that these are augmented by initiator protein binding. Here origin bending is examined by direct measurement of the ability of polymerized ori sequences to form small circles. The smallest circles observed (84 residues) are compatible with the required radius of curvature. Bend sites within the O protein binding sites, bend sites in the spacers between them, plus the inherent flexibility of non-bent DNA in the origin may all contribute to origin bending. The data also show that a bend site is required for O protein binding to DNA.     

Parallel association of Fos and Jun leucine zippers juxtaposes DNA binding domains

The protein products of the fos and jun proto-oncogenes form a heterodimeric complex that participates in a stable high affinity interaction with DNA elements containing AP-1 binding sites. The effects of deletions and point mutations in Fos and Jun on protein complex formation and DNA binding have been examined. The data suggest that Fos and Jun dimerize via a parallel interaction of helical domains containing a heptad repeat of leucine residues (the leucine zipper). Dimerization is required for DNA binding and results in the appropriate juxtaposition of basic amino acid regions from Fos and Jun, both of which are required for association with DNA.     

Static and initiator protein-enhanced bending of DNA at a replication origin

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, DNA packaging, and the recognition of specific DNA sequences by proteins. It has recently been demonstrated that DNA bending can be sequence-directed. Bent DNA has also been observed as a consequence of sequence-specific binding of proteins to DNA. In this report DNA of plasmid pT181 is shown to contain a bend at the replication origin. Furthermore, this bend is enhanced by the binding of the pT181 replication initiator protein, RepC, to the origin.     

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.     

Convergent solutions to binding at a protein-protein interface

The hinge region on the Fc fragment of human immunoglobulin G interacts with at least four different natural protein scaffolds that bind at a common site between the C(H2) and C(H3) domains. This "consensus" site was also dominant for binding of random peptides selected in vitro for high affinity (dissociation constant, about 25 nanomolar) by bacteriophage display. Thus, this site appears to be preferred owing to its intrinsic physiochemical properties, and not for biological function alone. A 2.7 angstrom crystal structure of a selected 13-amino acid peptide in complex with Fc demonstrated that the peptide adopts a compact structure radically different from that of the other Fc binding proteins. Nevertheless, the specific Fc binding interactions of the peptide strongly mimic those of the other proteins. Juxtaposition of the available Fc-complex crystal structures showed that the convergent binding surface is highly accessible, adaptive, and hydrophobic and contains relatively few sites for polar interactions. These are all properties that may promote cross-reactive binding, which is common to protein-protein interactions and especially hormone-receptor complexes.     

Heterogeneity of normal human diploid fibroblasts: isolation and characterization of one phenotype

Cultures of human diploid fibroblasts contain cells that respond to exposure to the first component of complement (C1) by initiating DNA synthesis and growth. The plasma membranes of these cells have specific binding sites for the C1q subcomponent of C1. A fluorescence-activated cell sorter was used to isolate a subset of cells with a high affinity for C1q, and the growth and synthesis activities of these high-affinity cells were studied after numerous replications in vitro. These cells synthesize DNA and grow faster than the parent cultures and low-affinity cells, and they produce two to three times as much protein. About 40 percent of their total protein synthesis activity is directed to collagen production, unusually high proportions of collagen types III and V being produced. These properties and the high affinity of the cells for C1q are retained for at least six cell transfers. This phenotype has the properties expected of fibroblasts in healing wounds and inflamed tissues.     

青鱼将DNA聚合酶β基因的电子克隆与分析

[目的]为了进行青鱼将DNA聚合酶β基因的电子克隆与分析的试验。[方法]利用生物信息学手段克隆了青鱼将DNA聚合酶β(OlPolβ)基因全长cDNA序列,并对其启动子和编码蛋白结构进行分析。[结果]该cDNA序列的全长1 716 bp,编码含338个氨基酸残基的蛋白。OlPolβ具有14个外显子和13个内含子。OlPolβ启动子上发现了SP1等一些转录因子的潜在结合位点。[结论]OlPolβ蛋白序列与其他脊椎动物的蛋白序列具有高度一致性。     

Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The structure of a 20-amino acid peptide inhibitor bound to the catalytic subunit of cyclic AMP-dependent protein kinase, and its interactions with the enzyme, are described. The x-ray crystal structure of the complex is the basis of the analysis. The peptide inhibitor, derived from a naturally occurring heat-stable protein kinase inhibitor, contains an amphipathic helix that is followed by a turn and an extended conformation. The extended region occupies the cleft between the two lobes of the enzyme and contains a five-residue consensus recognition sequence common to all substrates and peptide inhibitors of the catalytic subunit. The helical portion of the peptide binds to a hydrophobic groove and conveys high affinity binding. Loops from both domains converge at the active site and contribute to a network of conserved residues at the sites of magnesium adenosine triphosphate binding and catalysis. Amino acids associated with peptide recognition, nonconserved, extend over a large surface area.     

Activation of the cellular DNA damage response in the absence of DNA lesions

The cellular DNA damage response (DDR) is initiated by the rapid recruitment of repair factors to the site of DNA damage to form a multiprotein repair complex. How the repair complex senses damaged DNA and then activates the DDR is not well understood. We show that prolonged binding of DNA repair factors to chromatin can elicit the DDR in an ATM (ataxia telangiectasia mutated)- and DNAPK (DNA-dependent protein kinase)-dependent manner in the absence of DNA damage. Targeting of single repair factors to chromatin revealed a hierarchy of protein interactions within the repair complex and suggests amplification of the damage signal. We conclude that activation of the DDR does not require DNA damage and stable association of repair factors with chromatin is likely a critical step in triggering, amplifying, and maintaining the DDR signal.     

The periplasmic galactose binding protein of Escherichia coli

A specific high affinity galactose transport system called P(betag) can be induced by trace amounts of galactose in the medium by virtue of its own ability to capture and accumulate galactose. The transport system is coregulated with the production of a high affinity periplasmic galactose binding protein, which constitutes but one part of the transport system. Some transport negative mutants still remain producers of this binding protein. A close correlation exists between production of the active binding protein and the presence of galactose chemotaxis. The hypothesis, that this binding protein is a common element of the specific galactose transport system, P(betag), and of galactose chemotaxis is supported by observations on structural mutants, being defective in galactose binding protein as well as showing a lack of galactose chemotaxis. The binding protein is a monomer with two binding sites for galactose. Binding of one or two of the galactose molecules elicits specific conformational changes of the galactose binding protein (lowered affinity for galactose, increase of charges of the protein, increased fluorescence of tryptophan residues). The importance of these features for transport and for chemotaxis is discussed (70).     

The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses

The transition from the expression of alpha, the first set of five herpes simplex virus genes expressed after infection, to beta and gamma genes, expressed later in infection, requires the participation of infected cell protein 4 (alpha 4), the major viral regulatory protein. The alpha 4 protein is present in complexes formed by proteins extracted from infected cells and viral DNA fragments derived from promoter domains. This report shows that the alpha 4 protein forms specific complexes with DNA fragments derived from 5' transcribed noncoding domains of late (gamma 2) genes whose expression requires viral DNA synthesis as well as functional alpha 4 protein. Some of the DNA fragments to which alpha 4 binds do not contain homologs of the previously reported DNA binding site consensus sequence, suggesting that alpha 4 may recognize and interact with more than one type of DNA binding site. The alpha 4 proteins can bind to DNA directly. A posttranslationally modified form of the alpha 4 protein designated alpha 4c differs from the alpha 4a and alpha 4b forms with respect to its affinity for DNA fragments differing in the nucleotide sequences of the binding sites.     

Echinomycin binding sites on DNA

The preferred binding sites of echinomycin on DNA can be determined by a method called "footprinting." A 32P end-labeled restriction fragment from pBR322 DNA is protected by binding to echinomycin, and cleaved by a synthetic DNA cleaving reagent, methidiumpropyl--EDTA . Fe(II); the DNA cleavage products are then subjected to high-resolution gel analyses. This method reveals that echinomycin has a binding site size of four base pairs. The strong binding sites for echinomycin contain the central two-base-pair sequence 5'-CG-3'. From an analysis of 15 echinomycin sites on 210 base pairs of DNA, key recognition elements for echinomycin are contained in the sequences (5'-3') ACGT and TCGT (A, adenine; C, cytosine; G, guanine; T, thymine).     

Action at a distance along a DNA

A number of ways are known by which an event at one location on a DNA molecule can affect an event at a distant location on the same molecule. Three classes of mechanisms are described for such distal actions: tracking or translocation of a protein along a DNA, the association of two proteins bound at separate sites to form a DNA loop in between, and distal interactions that are affected by the topology of the DNA. The basic characteristics of each type of mechanism are discussed in terms of the known physicochemical properties of DNA. The various modes of action at a distance are often interrelated. Examples include the formation of positively and negatively supercoiled DNA loops by tracking and the strong effects of DNA topology on looping.