Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase

A functional immune system depends on the production of a wide range of immunoglobulin molecules. Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation. In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification. In hypermutating chicken DT40 B cells, most IgV mutations are dC --> dG/dA or dG --> dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase. Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC --> dT (and dG --> dA) transitions. Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance. This is good evidence that antibody diversification involves dC --> dU deamination within the immunoglobulin locus itself.     

Poly(dA).poly(dT) is a B-type double helix with a distinctively narrow minor groove

The structure of poly(dA).poly(dT) currently arouses great interest, mainly because dAn.dTn stretches are associated with considerable DNA bending. Until recently the heteronomous DNA described by Arnott et al., with the poly(dA) and poly(dT) chains in A and B conformations respectively, was the only detailed model of this structure. Following our earlier studies of the interaction of DNA and monovalent ions, we examined the X-ray diffraction of the bivalent Ca2+ salt of poly(dA).poly(dT) (Ca-poly(dA).poly(dT)) and found no sign of a heteronomous structure: Ca-poly(dA).poly(dT) in fibres shows fully equivalent B-type conformations of the opposite sugar-phosphate chains. A revision of the structure of the sodium salt, Na-poly(dA).poly(dT), based on this result, yields only a slightly heteronomous structure with each chain in a B-type conformation, which is in much better agreement with the experimental data underlying the original heteronomous model. Both structures, Ca- and Na-poly(dA).poly(dT), have a minor groove narrower than that of the B form: this peculiarity seems to be very important for the interaction of poly(dA).poly(dT) and biologically significant molecules (including proteins and antibiotics). The specific base-pair positions in poly(dA).poly(dT) may account for the DNA bending adjacent to dAn.dTn tracts.     

应用大肠杆菌的核苷磷酸化酶合成胸苷

通过大肠杆菌核苷磷酸化酶的转脱氧核糖基作用，从胸腺嘧啶和２‘－脱氧核苷合成胸苷。优化了以ｄＵ为底物合成胸苷的反应条件。以３０ｍｍｏｌ／Ｌ ｄＵ为底物其合成胸苷的转化率可达５．３％；以ｄＡ，ｄＣ，ｄＵ，ｄＧ的混合物为底物转化度为５２．６％；以ＤＮＡ降解后的２’－０脱氧核苷混合液作底物，反应液中胸苷浓度从９．２ｍｍｏｌ／Ｌ提高到１７．９ｍｍｏｌ／Ｌ。     

DNA bending at adenine . thymine tracts

Intrinsic bending of DNA molecules results from local structural polymorphism in regions of homopolymeric dA . dT which are at least 4 base pairs long; the A . T tracts must be repeated in phase with the helix screw. Bending, in the direction of base-pair tilt rather than roll, occurs at the junctions between the A . T tract and adjacent B-DNA, with a larger angle at the 3' than at the 5' end of the A tract.     

The structure of an oligo(dA).oligo(dT) tract and its biological implications

Poly(dA).poly(dT) has unusual properties in that it cannot associate into nucleosomes and short, phased runs of it cause DNA bending. The crystal structure of a B-type DNA dodecamer containing a homopolymeric run of six A.T base pairs shows that this region possesses special structural features, including a system of bifurcated hydrogen bonds, which explains some of the properties of this simple homopolymer.     

Measurement of anomalously high hydration of (dA)n.(dT)n double helices in dilute solution

Different DNA sequences have different physical properties, which seem to be important for their biological function. In particular, (dA)n.(dT)n has many unusual features, which include resistance to conformational changes in a variable chemical environment, an unusual thermodynamics of interaction with ligands, and the inability to reassociate into nucleosomes. Short A.T base-pair runs also play a critical role in DNA bending. It is believed that hydration of DNA is an important factor in determining the physical chemical and biological properties of different regions of DNA. Until now, however, it has not been possible to study the details of the hydration of DNA in dilute solution with sufficient sensitivity and precision. Moreover, it was not known if different base sequences differ in the extent of their hydration. Indirect evidence that (dA)n.(dT)n can be hydrated to a greater extent than other DNA sequences may be inferred from a recent study of the binding of drugs to polynucleotides. Here we used a novel high-precision technique measuring ultrasonic velocity to obtain direct estimates of the extent of hydration of various oligo- and polynucleotides in dilute solution. We report that different DNA sequences differ in their hydration, and that (dA)n.(dT)n in particular has an anomalously high level of hydration.     

Molecular spectroscopy: complexity of excited-state dynamics in DNA

Absorption of ultraviolet light by DNA is known to lead to carcinogenic mutations, but the processes between photon absorption and the photochemical reactions are poorly understood. In their study of the excited-stated dynamics of model DNA helices using femtosecond transient absorption spectroscopy, Crespo-Hernández et al. observe that the picosecond component of the transient signals recorded for the adenine-thymine oligonucleotide (dA)18.(dT)18 is close to that for (dA)18, but quite different from that for (dAdT)9.(dAdT)9; from this observation, they conclude that excimer formation limits excitation energy to one strand at a time. Here we use time-resolved fluorescence spectroscopy to probe the excited-state dynamics, which reveals the complexity of these systems and indicates that the interpretation of Crespo-Hernández et al. is an oversimplification. We also comment on the pertinence of separating base stacking and base pairing in excited-state dynamics of double helices and question the authors' assignment of the long-lived signal component found for (dA)18.(dT)18 to adenine excimers.     

AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification

After gene rearrangement, immunoglobulin variable genes are diversified by somatic hypermutation or gene conversion, whereas the constant region is altered by class-switch recombination. All three processes depend on activation-induced cytidine deaminase (AID), a B-cell-specific protein that has been proposed (because of sequence homology) to function by RNA editing. But indications that the three gene diversification processes might be initiated by a common type of DNA lesion, together with the proposal that there is a first phase of hypermutation that targets dC/dG, suggested to us that AID may function directly at dC/dG pairs. Here we show that expression of AID in Escherichia coli gives a mutator phenotype that yields nucleotide transitions at dC/dG in a context-dependent manner. Mutation triggered by AID is enhanced by a deficiency of uracil-DNA glycosylase, which indicates that AID functions by deaminating dC residues in DNA. We propose that diversification of functional immunoglobulin genes is triggered by AID-mediated deamination of dC residues in the immunoglobulin locus with the outcome--that is, hypermutation phases 1 and 2, gene conversion or switch recombination--dependent on the way in which the initiating dU/dG lesion is resolved.     

Importance of DNA stiffness in protein-DNA binding specificity

From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.     

三螺旋DNA分子久期方程的约化

利用矩阵分块技术和将二维矩阵化为一维矩阵的方法、明显地简化了poly(dT)·poly(dA)·poly(dT)的久期方程,大大节省了计算机内存空间和计算时间.这件方法可以用来计算具有螺旋对称性的所有巨分子的低频振动谱.     

装配式混凝土简支斜梁桥基频实用计算公式

以一座典型装配式混凝土简支梁桥为工程背景,采用数值分析方法开展斜交角、跨径、桥面宽度和横向扭转刚度对斜梁桥基频的影响分析.结果表明:随着斜交角增长,桥梁前三阶竖向振型对应频率增大;随斜梁桥跨径增大,结构整体竖向刚度降低,基频明显减小,减小速率趋缓;受斜梁桥弯扭耦合力学特征影响,基频随截面抗扭刚度增加显著提高.依据各关键参数对斜梁桥基频的影响分析结果,在现行规范桥梁基频估算公式的基础上,提出考虑截面抗扭刚度和斜交角的混凝土简支斜梁桥基频估算改进公式.通过多座实桥试验数据验证改进公式的精度和适用性.     

车用发电机定子绕组匝间短路故障下转子弯扭耦合振动特性分析

运用数值积分法,在建立发电机转子系统弯扭耦合振动模型基础上,考虑定子绕组匝间短路故障时发电机转子弯曲及扭转电磁刚度的影响,对不同程度匝间短路故障下转子的弯曲及扭转振动特性进行分析。结果表明,发电机定子匝间短路故障不仅会使转子弯曲和扭转振动加强,还会增加转子弯振和扭振中的倍频及高倍频成分;随短路程度加大,弯振的1、3、5等奇数次倍频振动量与扭振的2、4等偶数次倍频振动量逐渐增加,且弯振中1倍频振动量最大,扭振中2倍频振动量变化最为显著。     

儒教的家族观与后现代意义

白车身结构显著影响整车性能,而车身框架是车身设计的基础.在某MPV车型概念设计阶段,通过参数化技术,快速将车身主要框架划分为梁和接头结构.分别局部刚化处理49处左右对称结构,分析各结构对扭转刚度、弯曲刚度、后部扭转模态影响的灵敏度.结果表明,D柱下段、顶盖后横梁、下地板后纵梁等结构的单位质量对扭转刚度贡献量超过5%;后地板横梁、前地板后纵梁、后地板纵梁等结构单位质量对弯曲刚度的贡献量超过4%.通过8处灵敏度较高的结构优化和厚度优化,同时对性能影响小的结构减薄、减件,结合工艺改进,实现白车身质量不增加,扭转刚度提高9.0%,弯曲刚度提高4.8%,后扭模态提高2.0%.     

Sequence-directed curvature of DNA

DNAs from both prokaryotic and eukaryotic organisms have yielded restriction fragments which manifest markedly anomalous electrophoretic behaviour (reduced mobility) when run on polyacrylamide gels. We have shown previously that the abnormal electrophoretic behaviour of one such fragment is a consequence of stable curvature of the helix axis in solution. The molecules involved tend to contain oligo(dA)-oligo(dT) runs which are approximately in-phase with the helix repeat; however, the precise structural elements responsible for DNA curvature have not been identified. One popular model for curvature invokes a non-coplanar 'wedge-like' conformation of ApA/TpT dinucleotide pairs. Despite a lack of direct evidence in support of this model, it has been used to provide quantitative estimates of curvature. To critically evaluate the ApA wedge model, we have performed an electrophoretic analysis of a series of closely related DNA polymers in which oligo(dA)-oligo(dT) runs of different polarity were compared. We conclude that ApA dinucleotide wedges cannot account for DNA curvature. Therefore, quantitative estimates for ApA wedge deformations, based solely on apparent curvature, cannot be correct.     

中塔刚度对三塔缆索承重桥动力特性影响研究

为研究三塔缆索承重桥中塔刚度的合理取值,本文以马鞍山长江公路大桥和武汉二七长江大桥为例,探究中塔纵桥向弯曲刚度、横桥向弯曲刚度和扭转刚度与三塔悬索桥和三塔斜拉桥的动力特性的关系。研究表明:三塔缆索承重桥某些振型的频率受中塔刚度变化影响较大,并表现为振型频率随刚度放大而迅速增大,然后增大的趋势逐渐放缓,直到趋于稳定值。例如,三塔悬索桥中塔纵桥向弯曲刚度放大5倍时一阶反对称竖弯频率增大37.2%,而100倍时增大为38.0%。     

A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates

Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase eta (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD'2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase kappa, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10-15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the 'steric gate' residue of DinB (Phe13 --> Val) and that of its archaeal homologue Dbh (Phe12 --> Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.     

基于发动机轴系扭振抑制的扭弯减振器分析

扭弯复合减振器能同时抑制扭转振动和弯曲振动,降低发动机噪声.以发动机轴系扭转振幅最小化为目标,研究了扭弯复合减振器的设计方法,并分析扭振减振器或弯振减振器参数偏离对扭转振幅曲线的影响.分析表明:增大扭振减振器惯量比、降低弯振减振器惯量比,能减小扭转振幅曲线的共振峰值;弯振减振器阻尼比和刚度偏离最佳参数后,能增大曲线共振峰值,但影响程度很小;扭振减振器阻尼比和扭转刚度的偏离能显著增大峰值.     

Scanning tunnelling microscopy of Z-DNA

Scanning tunnelling microscopy (STM) has been used to map the surface topography of inorganic materials at the atomic level, and is potentially one of the most powerful techniques for probing biomolecular structure. Recent STM studies of calf thymus DNA and poly(rA).poly(rU) have shown that the helical pitch and periodic alternation of major and minor grooves can be visualized and reliably measured. Here we present the first STM images of poly(dG-me5dC).poly(dG-me5dC) in the Z-form. Both the general appearance of the fibres and measurements of helical parameters are in good agreement with models derived from X-ray diffraction.     

MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis.

Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type. Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate. This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.