The effect of ionising radiation and chemical methylation upon the activity and accuracy of E. COLI DNA polymerase I

The activity of $\text{E. coli}$ DNA polymerase I decreases on treatment with γ-rays, methylnitrosourea or dimethyl sulphate. In the case of the first two agents the decrease in activity is accompanied by a decrease in the accuracy of the enzyme in an $\text{in vitro}$ assay. There is no detectable change in the ratio of DNA polymerase activity to 3′→5′ exonuclease activity on treatment.     

DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen-binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference-mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.     

The in vitro interaction of the carcinogens, 4-nitroquinoline-N-oxide and its metabolite 4-hydroxylaminoquinoline-N-oxide, with intact rat liver mitochondria.

The inhibition of rat liver mitochondrial respiration caused by rotenone, is relieved by the 2 carcinogens, 4-nitroquinoline-N-oxide (NQO) and its metabolite 4-hydroxylaminoquinoline-N-oxide (HAQO). Thus these agents cause reducing equivalents to circumvent the first coupling site of the respiratory chain. This is another example of the experimental confluence between oxidative phosphorylation and chemical carcinogenesis.     

The effect of nicotinamide on unscheduled DNA synthesis in cultured hepatocytes

The synthesis of $\text{E. coli}$ proteins was examined, by two-dimensional O'Farrell gels, in mutant strains defective in all possible combinations of the RNA processing enzymes RNase III, RNase E and RNase P. We found that the synthesis of most proteins was unaffected; however, the synthesis of a significant number of proteins, 21 out of 80 tested, was drastically reduced in the strain defective in all three enzymes. It appears that the two enzymes RNase III and RNase E are responsible for most of these changes.     

The inhibitory effect of some lectins on the differentiation of Friend erythroleukemia cells

Growth and differentiation of Friend cells can be inhibited by treatment with concanavalin A and wheat-germ agglutinin. This inhibition is specific for cells that are in the early stages of the differentiation process (24–48 h after the addition of dimethyl sulfoxide) and is reversible upon treatment with the sugars competitive for these lectins. These results suggest a regulatory role for some plasma membrane-bound glycoproteins early in the differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide.     

DNA methylation is not necessary for the inactivation of the Tam3 transposon at non-permissive temperature in Antirrhinum

It has been proposed that DNA methylation plays an important role in the inactivation of transposons. This view stems from a comparison of the degree of methylation of transposons in the active and inactive state. However, direct evidence for the degree of methylation required for the suppression of transposition has not been reported. Transposon Tam3 in Antirrhinum majus undergoes somatic reversal of its transposition activity, which is tightly controlled by temperature: low temperature around 15 degrees C permits transposition, high temperatures around 25 degrees C strongly inhibits it. Our previous study had shown that the methylation state of the Tam3 end regions is negatively correlated with the Tam3 transposition frequency. The results of the present study reveal that the inactive state of Tam3 copies at high temperature is unlikely to be directly coupled to the methylation state. Treatment with methylation inhibitors (5-azacytidine or 5-azacytidine+ethionine) does not affect Tam3 excision frequency in calli derived from Antirrhinum hypocotyls. The results suggest that methylation is not essential for the suppression of Tam3 transposition at high temperature, but rather that some other mechanism(s) involved in the control of Tam3 transposition may be obscured by methylation.     

Specificity in carcinogen-DNA interaction: a theoretical exploration of the factors involved in the effect of neighboring bases on N-methyl-N-nitrosourea alkylation of DNA

Recent experimental studies indicate that in a polynucleotide chain neighboring bases have a significant effect on the relative alkylation of O6 or N7 of guanine by N-methyl-N-nitrosourea (MNU). This paper provides a theoretical exploration of this phenomenon in terms of an appropriate index of reactivity, called accessible surface integrated field (ASIF), introduced recently for the very sake of accounting for specificity or selectivity in drug-macromolecule interaction. The detailed analysis indicates that in the present case the observed variations in relative reactivity are attributable essentially to parallel variations in the accessibilities to the target atoms.     

A neutralizable epitope is induced on HGF upon its interaction with its receptor cMet

A new conformational neutralizable epitope is created on heptocyte growth factor (HGF), when it interacts with its receptor, cMet. By immunizing rabbits with HGF-cMet complex, we successfully generated a monoclonal antibody (SFN68) that inhibits HGF-cMet interaction, and blocks the biological function mediated by HGF. To define the epitope, we screened out an epitope-mimicking peptide, KSLSRHDHIHHH, from a phage display of combinatorial peptide library. In molecular mimicry this peptide bound to cMet and inhibited HGF-cMet interaction. No humoral response was induced to this epitope-mimicking peptide when immunization was done with HGF alone.     

The presence of terminal deoxynucleotidyl transferase in the N-methyl-N-nitrosourea induced leukaemia in BDF1 mice and its effect on the accuracy of the DNA polymerases.

DNA polymerases have been prepared from leukaemic and normal spleens and their fidelity in copying a polyd AT).polyd(AT) template assessed. The leukaemic cytoplasmic DNA polymerases were less accurate than the controls whereas no difference in accuracy was observed in the nuclear DNA polymerases. The preparations of leukaemic cytoplasmic DNA polymerase also contained the enzyme terminal deoxynucleotidyl transferase. When this enzyme was removed by further purification the accuracy of the cytoplasmic DNA polymerases increased to that of the controls.     

Hinge-mediated dimerization of SMC protein is essential for its dynamic interaction with DNA

Structural maintenance of chromosomes (SMC) proteins play central roles in regulating higher order chromosome dynamics from bacteria to humans. As judged by electron microscopy, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel, coiled-coil arms with a flexible hinge. Site-directed cross-linking experiments show here that dimerization of BsSMC is mediated by a hinge-hinge interaction between self-folded monomers. This architecture is conserved in the eukaryotic SMC2-SMC4 heterodimer. Analysis of different deletion mutants of BsSMC unexpectedly reveals that the major DNA-binding activity does not reside in the catalytic ATPase domains located at the ends of a dimer. Instead, point mutations in the hinge domain that disturb dimerization of BsSMC drastically reduce its ability to interact with DNA. Proper hinge function is essential for BsSMC to recognize distinct DNA topology, and mutant proteins with altered hinge angles cross-link double-stranded DNA in a nucleotide-dependent manner. We propose that the hinge domain of SMC proteins is not a simple dimerization site, but rather it acts as an essential determinant of dynamic SMC-DNA interactions.     

The effect of the cryoprotectants,dimethylsulfoxide and glycerol on water transport in the human red blood cell

The response of human red blood cells to the cryoprotective agents, DMSO and glycerol, has been investigated using a pulsed NMR method. The experimentally determined parameters are: (1) the intracellular transverse relaxation time, T_2a; (2) the mean residence time of intracellular water, τ_a, which is effectively a reciprocal measure of the rate of water transport across the red blood cell membrane; and (3) the activation energy for this process. The quantitative data indicate that the observed effects are colligative rather than species-specific in origin.     

哺乳动物DNA甲基化及其在体细胞诱导重编程中的作用

诱导多能干细胞(Induced pluripotent stem cell, iPS)技术提供了将终末分化的细胞逆转为多潜能干细胞的可能, 在干细胞基础理论研究和再生医学中具有重要意义。然而, 目前体细胞诱导重编程方法效率极低, 常发生不完全的重编程。研究表明, 在不完全重编程的细胞中存在体细胞的表观遗传记忆, 而DNA甲基化作为相对长期和稳定的表观遗传修饰, 是影响重编程效率和iPS细胞分化能力的重要因素之一。哺乳动物DNA甲基化是指胞嘧啶第五位碳原子上的甲基化修饰, 常发生于CpG位点。DNA甲基化能够调节体细胞特异基因和多能性基因的表达, 因此其在哺乳动物基因调控、胚胎发育和细胞重编程过程中发挥着重要作用。此外, 异常DNA甲基化可能导致iPS细胞基因印记的异常和X染色体的失活。文章重点围绕DNA甲基化的机制、分布特点、及其在体细胞诱导重编程中的作用进行了综述。     

The interaction of emetine with DNA and its effect on the adsorption of certain bacteriophages

The interaction of emetine with DNA in solution at low ionic strength did not bring about a detectable change of the spectrum of either DNA or emetine in the ultraviolet region but resulted in stabilization of the secondary structure of DNA. The irreversible interaction of emetine with DNA or bacteriophage coat, which would result in a loss of infectivity of the lambda cI 857, T7 or T4 BOI bacteriophages did not take place. However, emetine decreased the adsorption of the bacteriophage lambda cI 857 onEscherichia coli C 600, probably due to interaction with cellular receptors.     

Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2alpha, a truncated form of nectin-2alpha incapable of interacting with afadin (nectin-2alpha-DeltaC), or a point-mutated form of nectin-2alpha capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2alpha. We found that the interaction of nectin-2alpha with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2alpha-DeltaC showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2alpha with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2alpha was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.     

Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation

Postreplicative maintenance of genomic methylation patterns was proposed to depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core component of the replication machinery. We investigated how the slow and discontinuous DNA methylation could be mechanistically linked with fast and processive DNA replication. Using photobleaching and quantitative live cell imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in living cells showed that this interaction accounts for a 2-fold increase in methylation efficiency. Expression of this mutant in mouse dnmt1^−/− embryonic stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with the replication machinery enhances methylation efficiency, but is not strictly required for maintaining global methylation. The transient nature of this interaction accommodates the different kinetics of DNA replication and methylation while contributing to faithful propagation of epigenetic information.