中国人结直肠癌患者碱基切割修复基因胚系突变及与腺瘤样息肉基因体细胞突变的关系研究

取42例经过病理确认的结直肠癌患者癌组织及癌旁正常组织,抽提基因组DNA,应用单链构象多态性分析(SSCP)、变性高效液相色谱(DHPLC)分析,结合DNA直接测序,在全基因分析humanmutY homologue(MYH)基因胚系突变的同时,探讨其对结直肠癌细胞adenomatous polyposis coli(APC)基因体细胞突变的影响.结果42例患者中检出6例(14.29%)携带MYH基因的胚系突变,其中3例(7.14%)为MYH基因第2外显子单体型突变c.53C>T/c.74G>A(p.Pro18Leu/p.Gly25 Asp),3例(7.14%)为第12外显子的单碱基替换导致的错义突变c.972G>C(p.Gln324His).初步分析显示,这两种突变在正常对照组的检出较低,仅为3/213(1.41%)和0/59(0%).另一方面4/6例携带MYH基因胚系突变患者的癌组织样本中检出5个APC基因体细胞突变.本文结果提示,散发性结直肠癌患者中频繁检出MYH基因的胚系突变,其存在可能导致细胞DNA氧化损伤修复功能减弱,并使机体细胞APC基因发生突变的风险增高,从而可能参与部分结直肠癌的发生.     

丙二醇和吐温-80对人淋巴细胞的SCE的诱发及DNA损伤

本文研究了医药工业中常用的两种助溶剂丙二醇和吐温-80对人淋细胞姊妹染色单体互换(SCE)的诱发作用及对 DNA 的损伤作用.丙二醇(4.13mg/mL)对 SCE 无明显的诱发作用,对 DNA 复制模板也无损伤.吐温-80能诱发 SCE 频率增高,剂量与效应呈线性相关.当浓度为0.001～0.2mg/mL 时与对照组相比差异极显著(p<0.01).使 SCE 频率增加到对照组的2倍的浓度为0.12mg/mL.浓度为1mg/mL 时严重抑制细胞增殖和分裂.它也能损伤 DNA 复制模板.因此,用吐温-80作助溶剂时应该慎重.     

真菌中DNA重复序列诱导点突变的研究进展

1987年,由Selker等在粗糙脉孢菌中首次发现重复序列诱导点突变(repeat-induced point mutation,RIP).在重复序列诱导点突变过程中,搜寻前减数分裂组织单倍体核中DNA的重复序列,然后发生众多的碱基C到T的突变,产生富碱基T+A片段,从而使重复序列中的G-C碱基对发生转换突变成为A-T碱基对.此外,发生RIP的序列多集中在着丝粒区域,主要是转座子甲基化后的遗迹.移动转座子是真核生物基因组进化的主要驱动力.对于真菌,重复序列诱导点突变(RIP)在减数分裂过程中通过突变多拷贝DNA,能最大限度地减少转座子的影响,因此对RIP的研究在一定程度上能有助于了解基因组进化的真谛.综述了重复序列诱导点突变的产生机制,以及真菌中重复序列诱导点突变的研究进展.     

DNA化学损伤的电化学检测

0 引言 DNA是重要的遗传物质,在机体的生长过程中,它不可避免地受到各种因素的作用而产生损伤,从而直接影响DNA的复制、转录和蛋白质的合成,进而影响细胞生长、发育、遗传、代谢和繁殖等生命基本过程,还会造成细胞突变、癌变、老化甚至死亡.因此,对DNA损伤及其检测方法的研究不仅有助于更好地了解生命体的基本过程,而且还可为疾病的临床诊断、治疗甚至新的基因治疗药物的发现提供理论基础.     

细胞自噬与高迁移率族蛋白B-1(HMGB1)介导肿瘤耐药的研究进展

恶性肿瘤可通过多种细胞机制,产生对抗癌药物和放疗的抗性,即所谓耐药现象.细胞自噬是肿瘤细胞耐药的一个重要原因.高迁移率族蛋白B-1(HMGB1)是高度保守的非组蛋白DNA结合蛋白,在DNA结构、基因转录、基因重组、DNA损伤修复以及细胞存活等方面发挥重要的调控作用;HMGB1在细胞内的功能,与其氧化还原状态和细胞定位息...     

纤细裸藻褪色突变株的残留质体及其对核基因表达的影响

用氧氟沙星(Ofl)和链霉素(Sm)分别处理纤细裸藻获得褪色突变株.电子显微镜观察显示细胞中存在残留质体,其中一个Ofl突变株质体有原初类囊体膜形成,而两个Sm突变株质体内有异常致密、发达的膜结构.用PCR方法检查了质体DNA的9个基因,显示所有突变株均有核糖体蛋白基因丢失,其中一个Sm突变株仍保留质体DNA的大部分基因,其余突变株质体DNA则基本丢失.通过差异显示和RT-PCR方法证明叶绿体的退化在完全黑暗异养条件下也可导致某些核基因转录的变化.     

竹红菌素研究的进展

竹红菌素(Hypocrellin)是在我国最早发现的一种新型光敏剂.它的光生物活性可抑制细菌生长.近几年的研究表明,细胞吸收后,它主要分布于细胞膜及胞浆中,只有少量的进入细胞核中.竹红菌素加光照后对细胞具有明显的杀伤作用,使细胞存活率显著下降.竹红菌素光敏作用靶部位是细胞膜,线粒体和微粒体是其敏感靶部位,而光敏作用引发的脂自由基等产物可能是诱导DNA等损伤,最终导致细胞死亡的重要原因.     

锌指核酸酶技术最新进展及其在植物基因工程中的应用

锌指核酸酶(zinc finger nucleases,ZFNs)是近年来发展起来的一项定点改变基因组序列的新技术.它能够特异切开基因组中的特定序列,产生双链断裂DNA,诱导细胞启动DNA损伤修复过程,在修复过程中实现对靶基因的敲除或改变.这一技术可以通过删除某一基因或一个基因的一部分、导入点突变等,对该基因的功能进行研究,也可以定向改变基因组序列,使生物体获得新的功能.这项技术在基因功能的研究和新药的研发、新品种选育等方面有着十分广阔的应用前景,是后基因组时代基因功能研究的重要手段.介绍了ZFNs工作原理,重点阐述了该技术近几年的最新进展及其在植物学领域的应用,同时也对ZFNs存在的问题进行了深入分析.     

DNA拓扑异构酶Ⅱβ结合蛋白1（TopBP1）磷酸化功能位点推测及功能分析

为阐明DNA拓扑异构酶Ⅱβ结合蛋白1(TopBP1)参与DNA损伤修复应答的分子机制，本研究通过生物信息学分析，发现多个潜在的TopBP1磷酸化位点T860，S887，T1104及T1167，利用分子生物学手段从人cDNA文库中扩增并获得TopBP1克隆质粒，将上述磷酸化位点突变为丙氨酸，观察突变质粒转染细胞对DNA损伤修复的应答反应. 结果显示，在粒子射线照射或化疗药物处理细胞后，第1104丙氨酸突变（T1104A） 的TopBP1蛋白导致pRPA32-S33的磷酸化水平大幅降低，同时细胞周期检验点失活，严重阻滞了DNA应激反应，证实T1104是TopBP1参与DNA损伤修复的关键活性位点.     

紫外辐射诱导植物细胞DNA损伤的彗星检测

单细胞凝胶电泳(彗星检测)已广泛应用于动物细胞DNA损伤检测.该文报道了单细胞凝胶电泳应用于UV-B诱导植物细胞DNA损伤的检测.通过对动物细胞的单细胞凝胶电泳实验方法的改进,获得了在植物细胞中应用的最佳条件.以植物细胞原生质体为材料,并结合T4 Endonu lease V的运用,显著提高了UV-B诱导植物细胞DNA损伤提高彗星检测的敏感性和特异性.植物细胞DNA损伤的彗星图像经CASP软件处理并量化,结果表明:UV-A和UV-B均能诱导发生DNA单链断裂;UV-A在一定的剂量下诱导少量嘧啶二聚体的形成,而UV-B则强烈诱导嘧啶二聚体的产生.     

Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions

DNA lesions can often block DNA replication, so cells possess specialized low-fidelity, and often error-prone, DNA polymerases that can bypass such lesions and promote replication of damaged DNA. The Saccharomyces cerevisiae RAD30 and human hRAD30A encode Pol eta, which bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Here we show that a related human gene, hRAD30B, encodes the DNA polymerase Pol iota, which misincorporates deoxynucleotides at a high rate. To bypass damage, Pol iota specifically incorporates deoxynucleotides opposite highly distorting or non-instructional DNA lesions. This action is combined with that of DNA polymerase Pol zeta, which is essential for damage-induced mutagenesis, to complete the lesion bypass. Pol zeta is very inefficient in inserting deoxynucleotides opposite DNA lesions, but readily extends from such deoxynucleotides once they have been inserted. Thus, in a new model for mutagenic bypass of DNA lesions in eukaryotes, the two DNA polymerases act sequentially: Pol iota incorporates deoxynucleotides opposite DNA lesions, and Pol zeta functions as a mispair extender.     

Two levels of protection for the B cell genome during somatic hypermutation

Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and completed by error-prone processing of the resulting uracils by mismatch and base excision repair factors. Somatic hypermutation represents a threat to genome integrity and it is not known how the B cell genome is protected from the mutagenic effects of somatic hypermutation nor how often these protective mechanisms fail. Here we show, by extensive sequencing of murine B cell genes, that the genome is protected by two distinct mechanisms: selective targeting of AID and gene-specific, high-fidelity repair of AID-generated uracils. Numerous genes linked to B cell tumorigenesis, including Myc, Pim1, Pax5, Ocab (also called Pou2af1), H2afx, Rhoh and Ebf1, are deaminated by AID but escape acquisition of most mutations through the combined action of mismatch and base excision repair. However, approximately 25% of expressed genes analysed were not fully protected by either mechanism and accumulated mutations in germinal centre B cells. Our results demonstrate that AID acts broadly on the genome, with the ultimate distribution of mutations determined by a balance between high-fidelity and error-prone DNA repair.     

A model for SOS-lesion-targeted mutations in Escherichia coli

The UmuD'2C protein complex (Escherichia coli pol V) is a low-fidelity DNA polymerase (pol) that copies damaged DNA in the presence of RecA, single-stranded-DNA binding protein (SSB) and the beta,gamma-processivity complex of E. coli pol III (ref. 4). Here we propose a model to explain SOS-lesion-targeted mutagenesis, assigning specific biochemical functions for each protein during translesion synthesis. (SOS lesion-targeted mutagenesis occurs when pol V is induced as part of the SOS response to DNA damage and incorrectly incorporates nucleotides opposite template lesions.) Pol V plus SSB catalyses RecA filament disassembly in the 3' to 5' direction on the template, ahead of the polymerase, in a reaction that does not involve ATP hydrolysis. Concurrent ATP-hydrolysis-driven filament disassembly in the 5' to 3' direction results in a bidirectional stripping of RecA from the template strand. The bidirectional collapse of the RecA filament restricts DNA synthesis by pol V to template sites that are proximal to the lesion, thereby minimizing the occurrence of untargeted mutations at undamaged template sites.     

Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.     

体外定向分子进化的新策略及新应用

体外定向分子进化是发现和改造生物活性分子的重要方法,提供了一种高效的获得多样性的方法。DNA改组(DNA shuffling)是重要的体外分子进化技术,结合高通量筛选能够改造许多重要的医药、工业、环境保护等方面的商业酶。近年来,许多体外分子进化的新策略和新方法层出不穷,得到了良好的发展和应用,其中有代表性的11种是DNA家族改组(DNA family shuffling),部分基因片段改组、单链DNA家族改组(SSDNAs)、简并引物基因改组(DOGS)、基因组改组(Genome Shuffling)、瞬时模板的随机嵌合(RACHITT)、单向引物的随机重组(MURA)、自我复制(CSR)改组、易错环行扩增(error-prone RCA)、基于遗传密码随机切除(COBARDE)、核酸内切酶V(endonuclease V)替代核酸内切酶DNaseI等。     

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.     

Role of poly(ADP-ribose) formation in DNA repair.

The abundant nuclear enzyme poly(ADP-ribose) polymerase catalyses the synthesis of poly(ADP-ribose) from nicotinamide adenine dinucleotide (NAD+). This protein has an N-terminal DNA-binding domain containing two zinc-fingers, which is linked to the C-terminal NAD(+)-binding domain by a short region containing several glutamic acid residues that are sites of auto-poly(ADP-ribosyl)ation. The intracellular production of poly(ADP-ribose) is induced by agents that generate strand interruptions in DNA. The branched homopolymer chains may attain a size of 200-300 residues but are rapidly degraded after synthesis. The function of poly(ADP-ribose) synthesis is not clear, although it seems to be required for DNA repair. Here we describe a human cell-free system that enables the role of poly(ADP-ribose) synthesis in DNA repair to be characterized. The results indicate that unmodified polymerase molecules bind tightly to DNA strand breaks; auto-poly(ADP-ribosyl)ation of the protein then effects its release and allows access to lesions for DNA repair enzymes.