8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.     

Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis

The expression of the Escherichia coli DNA polymerases pol V (UmuD'2C complex) and pol IV (DinB) increases in response to DNA damage. The induction of pol V is accompanied by a substantial increase in mutations targeted at DNA template lesions in a process called SOS-induced error-prone repair. Here we show that the common DNA template lesions, TT (6-4) photoproducts, TT cis-syn photodimers and abasic sites, are efficiently bypassed within 30 seconds by pol V in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol III's processivity beta,gamma-complex. There is no detectable bypass by either pol IV or pol III on this time scale. A mutagenic 'signature' for pol V is its incorporation of guanine opposite the 3'-thymine of a TT (6-4) photoproduct, in agreement with mutational spectra. In contrast, pol III and pol IV incorporate adenine almost exclusively. When copying undamaged DNA, pol V exhibits low fidelity with error rates of around 10(-3) to 10(-4), with pol IV being 5- to 10-fold more accurate. The effects of RecA protein on pol V, and beta,gamma-complex on pol IV, cause a 15,000- and 3,000-fold increase in DNA synthesis efficiency, respectively. However, both polymerases exhibit low processivity, adding 6 to 8 nucleotides before dissociating. Lesion bypass by pol V does not require beta,gamma-complex in the presence of non-hydrolysable ATPgammaS, indicating that an intact RecA filament may be required for translesion synthesis.     

VIP and noradrenaline act synergistically to increase cyclic AMP in cerebral cortex

There is growing evidence that two, or possibly more, neurotransmitters can coexist within the same neurone. In particular, the presence of a peptide and a biogenic amine has been demonstrated in the same terminals of central and peripheral neurones. These findings have led to the hypothesis that neurotransmitters, coexisting within the same neurones, can interact at pre- or postsynaptic sites in a functionally coordinated manner. However, interactions between neurotransmitters contained in distinct neuronal systems terminating within the same region of the central nervous system (CNS) can be envisaged. We have examined this last possibility in the cerebral cortex, an area of the CNS where the two neurotransmitters vasoactive intestinal polypeptide and noradrenaline are contained in separate neuronal systems and where they both stimulate the formation of cyclic AMP. We report here that vasoactive intestinal polypeptide and noradrenaline act synergistically to stimulate the formation of cyclic AMP and that this synergistic interaction is antagonized by the specific alpha-adrenergic antagonist phentolamine.     

Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions

DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.     

DNA损伤修复蛋白在胆囊病变组织中的表达及其临床意义

目的 研究胆囊腺癌、癌旁组织、腺瘤性息肉和慢性胆囊炎组织中DNA损伤修复蛋白hMSH2和hMLH1表达及其临床意义.方法 选取胆囊腺癌108例、癌旁组织46例、腺瘤性息肉15例和慢性胆囊炎35例手术切除标本常规作石蜡包埋切片,采用EnVisionTM免疫组化法检测hMSH2和hMLH1.结果 hMSH2和hMLH1表达阳性率,胆囊腺癌分别为50.0%和49.1%、评分分别为2.2±1.9和2.2±1.8,均明显低于癌旁组织（阳性率分别为84.8%和87.0%;评分分别为3.9±1.3和4.2±1.2）、腺瘤性息肉（阳性率分别为80.0%和86.7%;评分分别为3.7±1.3和4.0±1.1）及慢性胆囊炎组织（阳性率分别为88.6%和88.6%;评分分别为4.1±1.1和3.9±1.1）（P＜0.05）;不同类型良性病变中2种DNA损伤修复蛋白表达阳性率及其评分均无明显差异（P＞0.05）.hMSH2和hMLH1表达阴性的良性病变胆囊上皮均呈中至重度不典型增生的病理形态学表现.腺瘤癌变或高分化腺癌、肿块最大径＜2 cm,无淋巴结转移及未侵犯周围组织的病例hMSH2和hMLH1表达阳性率及其评分均明显地高于低分化腺癌、肿块最大径≥2 cm、淋巴结转移及侵犯周围组织病例（P＜0.05）;2种DNA损伤修复蛋白表达与患者性别、年龄及有无胆囊结石均无明显关系（P＞0.05）.结论 DNA损伤修复蛋白表达水平均为反映胆囊腺癌发生、进展、临床生物学行为及预后的重要标记物,检测其表达水平对指导预防和早期发现、临床化疗胆囊癌可能有一定临床意义.     

Folding DNA to create nanoscale shapes and patterns

'Bottom-up fabrication', which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex).     

微卫星DNA技术及其应用的探讨

微卫星DNA是一类短串联重复的寡核苷酸，广泛的分散于整个基因组中，具有多态性高、突变速率快等特点，在医学领域、生物学领域应用广泛。本文综述了微卫星多态性形成机制、序列获得方法及其应用途径，尤其是微卫星不稳定性与疾病的相互关系。     

天冬氨酸-邻二氮菲-Cu(Ⅱ)配合物与DNA相互作用及其对DNA的降解

利用循环伏安法研究天冬氨酸-邻二氮菲-Cu(Ⅱ)配合物([Cu(Phen)(Asp)]2+)与脱氧核糖核酸(DNA)之间的相互作用.结果表明,在pH6.8的磷酸盐缓冲溶液中,配合物[Cu(Phen) (Asp)]2+产生一对灵敏的氧化还原峰,峰电流随DNA浓度的增大而逐渐降低.在最佳实验条件下,测定线性范围为0.02-10.05 μg/mL,检出限为0.002 μg/mL,且研究该配合物存在条件下对DNA的电化学诱导降解.     

一种简便高效提取细菌总DNA和RNA的方法

本科实验教学中,细菌总DNA和RNA的提取是一个基础实验.但该实验中经常出现DNA提取不出来、产量低、条带不清晰、RNA降解、蛋白残留多等问题,影响了学生对细菌DNA和RNA的直观认识,降低了实验教学效果.将常规提取方法进行精简优化,摸索出一种简便、稳定、高效、成功率高的DNA和RNA同时提取的方法.所获样品电泳条带清晰,无降解,蛋白残留少;且实验步骤少,基本每个学生都能获得理想结果,明显改善了教学实验效果,值得在本科生物化学实验教学中推广.     

改进的DNA遗传算法在指派问题中的应用

提出了一种基于优秀基因片段思想的DNA遗传算法,将这段基因片段提取出来并将它遗传到后代中,可以加快收敛速度.给出了DNA遗传算法的结构,讨论了选择、交叉和变异算子的具体操作,并将其运用到指派问题最优解的求解中,给出了具体的实现方法.仿真实验验证了算法的有效性和实用性.     

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。     

一种DNA侧翼序列分离技术--TAIL-PCR

在分子生物学研究中，基因克隆和分子杂交的探针制备等操作常需分离与已知DNA序列邻近的未知序列，热不对称交错PCR(简称TAIL—PCR)反应技术能够较好地解决上述难题。该技术通过3个嵌套的特异性引物分别和简并引物组合进行连续的PCR循环，利用不同的退火温度选择性地扩增目标片段，所获得的片段可以直接用做探针标记和测序模板。TAIL—PCR技术简单易行，反应高效灵敏，产物的特异性高，重复性好，能够在较短的时间内获得目标片段，已经成为分子生物学研究中的一种实用技术。笔综述了TAIL-PCR反应的原理、引物设计、反应条件设置及产物分析。     

六氯苯暴露导致梨形环棱螺脂质过氧化及DNA损伤研究

采用暴露实验方法,研究了不同浓度HCB(分别为0.0、2.0、4.0、8.0、16.0 mg/L)在不同暴露时间(0-14d)下对梨形环棱螺肝脏和鳃中MDA、ROS含量以及DNA单链断裂程度的影响。结果表明:HCB对梨形环棱螺肝脏和鳃中MDA、ROS含量和DNA断裂均有明显影响。肝脏和鳃的MDA含量在HCB暴露过程中波动升高,直到在第4天或第7天达到峰值,然后MDA含量持续下降,到第14天时,除了16.0mg/L剂量组与对照组无显著差异外,其他剂量组MDA仍处于显著被诱导状态。ROS水平随暴露时间和剂量增加而表现出升高的趋势,并且高剂量组较中低剂量组更早达到峰值,随着暴露时间的进一步延长,各剂量组ROS含量均有一定程度的下降,但在实验结束时仍然显著高于对照组。各剂量组DNA在HCB暴露起始时,出现一定程度的单链断裂现象,即F值迅速降低,随后F值有所上升,在第4天时F值达到最高点,显示DNA有一定程度的修复效应。接着F值呈现出持续下降的趋势,并且高剂量组的DNA单链断裂明显,表现出时间-剂量效应。结果提示,上述生物标志物在六氯苯对梨形环棱螺进行毒性暴露过程中的变化较为敏感,可以作为指示六氯苯对梨形环棱螺毒理效应的生物标志物。