分枝杆菌非典型错配修复及其在抗生素耐药中的研究进展

错配修复(mismatch repair, MMR)是生物体DNA复制后的一种常见修复系统，对于维持基因组稳定性至关重要，其关键步骤由MutS和MutL蛋白家族的成员执行，尽管这种修复途径十分重要，但在许多古菌和放线菌基因组中并不存在MutS或MutL的同源蛋白。这类细菌(例如分枝杆菌等)采用另一种非典型的MMR途径，由核酸内切酶EndoMS/NucS发挥关键作用，与典型MMR蛋白(MutS/MutL)相比没有结构同源性。EndoMS/NucS介导的非典型错配修复在分枝杆菌DNA修复、突变和同源重组以及抗生素耐药等方面发挥重要作用。本文通过对比典型MMR途径和非典型MMR途径，深入阐述了分枝杆菌EndoMS/NucS介导的非典型MMR途径及其最新进展，以期为分枝杆菌错配修复分子机制带来新见解以及对分枝杆菌抗生素治疗提供研究线索。     

错配识别蛋白MutS的研究及应用进展

错配修复(mismatchrepairsystem,MMR)系统维护着遗传物质的稳定性。错配识别蛋白MutS是错配修复系统行使修复功能的第一个蛋白,具有识别并结合错配的能力。MutS蛋白具有特异性结合错配的特殊功能,在检测突变和SNP的研究中具有很大的应用潜力。近年来已有一些报道介绍了Muts蛋白的一些方法,虽然这些方法还有待改进,但MutS应用前景仍然十分诱人。     

Mechanisms and functions of DNA mismatch repair

DNA mismatch repair （MMR） is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including hereditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.     

DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.     

错配修复蛋白MutS的新功能位点

MutS蛋白是DNA错配修复系统的关键成份,其突变会使细胞失去正常的错配修复功能,导致基因组不稳定和细胞异常.本研究利用易错PCR随机突变和利福平筛选,建立了研究MutS蛋白的新方法,发现影响MutS错配修复功能的新位点,并利用表面等离子共振、分子筛、farwestern等方法对错配修复功能缺陷的突变体进行了活性测定和分析;通过揭示MutS与错配修复功能相关的新信息,为MutS同源物多态性的研究及人源MutS同源物突变与癌症相关的研究提供新的线索.     

DNA错配修复系统组成和功能的研究进展

DNA错配修复(Mismatch repair,MMR)系统广泛的存在于从原核到真核的生物体中,是进化上保守的生化通路.MMR系统由一系列特异性修复DNA碱基错配的酶分子(错配修复基因产物)组成.细胞由于此系统的存在使DNA复制保持忠实性,从而保持遗传物质的完整性和稳定性,避免遗传物质发生突变.MMR系统基因的失活会导致自发突变率的明显增加,从而导致微卫星不稳定(MSI),可能引发某些肿瘤发生.近年来,MMR系统的研究越来越受到学者的重视,对MMR作用机制及组成该系统的几种酶蛋白结构与功能方面的研究不断深入,加深了对MMR系统的理解.这些为MMR系统相关的应用研究,尤其是为肿瘤发生奠定了理论的基础.本文重点讨论了错配修复系统的蛋白组成、各蛋白的功能及它们如何相互协调发挥作用的最新研究进展.     

MutL融合蛋白的高效表达及其伴侣功能研究(英文)

DNA错配修复蛋白MutL和其它的修复蛋白相互作用来共同完成大肠杆菌甲基介导的错配修复过程 .为研究修复蛋白MutL的体外生物功能构建了融合蛋白Trx His6 Linkerpeptide MutL(THLL)的表达载体并使其高效表达及易于纯化 .mutL基因片段是以E .coliK 12基因组为模板经PCR扩增获得 ,并通过基因的体外拼接成功构建了融合蛋白THLL表达载体pET32a linkerpeptide mutL .重组菌株E .coliAD4 94 (DE3) pET32a linkerpeptide mutL经过IPTG的诱导表达了融合蛋白THLL .收集菌体细胞、超声波破碎后离心取上清进行SDS PAGE分析 ,结果表明有一与预期分子量(84kD)相应的诱导表达条带出现 ,其表达量约占全细胞蛋白的 30 %且以可溶形式存在 .利用固定化金属离子 (Ni2 +)配体亲和层析柱纯化融合蛋白THLL ,其纯度达到 90 % .通过非变性凝胶电泳分析 ,对融合蛋白THLL在DNA错配修复过程中的分子伴侣生物功能进行了系统研究 .结果表明 ,THLL能增加融合蛋白Trx His6 Linkerpeptide MutS (THLS)与含有错配碱基DNA双链的结合 ,但受ATP的浓度变化影响很大     

重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响

毕赤酵母是目前最优秀的外源蛋白表达系统之一。本文着重对重组毕赤酵母甲醇利用表型（Mut+型、MutS型和Mut-型）、基因剂量对外源蛋白高效表达的影响机理进行综述。MutS型的比生长速率和蛋白产率比Mut+型低、发酵周期长、副产物（如乙醇、乙酸等）形成速率不同。外源基因拷贝数对外源蛋白的影响主要有三种情况：（1）高基因拷贝数对外源蛋白表达水平有明显的正效应作用;（2）基因拷贝数增加反而降低了表达水平,即负效应作用;（3）重组蛋白表达与基因剂正相关,之后则表现负相关关系,这可能与外源蛋白翻译后加工有关（如二硫键形成、折叠等）,而与分子伴侣共表达可促进外源蛋白的高表达。     

大肠杆菌细胞裂解系统的构建及其在真菌毒素降解酶表达中的应用

目的:大肠杆菌中分泌表达重组蛋白受限于其分泌效率,为此设计构建大肠杆菌诱导裂解系统以实现胞内重组蛋白的快速高效分泌。方法:利用大肠菌素E7对细胞的裂解能力,构建共表达目标重组蛋白和E7的大肠杆菌细胞裂解系统,使目标重组蛋白在E7表达后得以释放到培养基中。结果:首先以红色荧光蛋白(red fluorescent protein,RFP)为报告基因,在pET28a(+)载体上构建大肠杆菌素E7和红色荧光蛋白两个表达盒,通过对比分析IPTG一步诱导和IPTG-阿拉伯糖分步诱导系统蛋白质的表达效果,发现分步诱导系统能够更高效地表达并释放目标蛋白到培养基。在IPTG-阿拉伯糖分步诱导裂解系统中表达玉米赤霉烯酮降解酶基因,培养基上清液中检测到玉米赤霉烯酮降解酶有较好的表达量和较高的活性,能够在37℃反应30min的条件下降解约5. 8μg玉米赤霉烯酮毒素。结论:利用大肠菌素E7成功构建大肠杆菌细胞裂解系统,并且此系统在快速释放胞内表达外源蛋白方面有适用性。     

信号肽在大肠杆菌分泌系统中的研究与应用进展

大肠杆菌以其明显的优势成为表达重组蛋白常用的系统,但是大肠杆菌本身不具备细胞内形成二硫键的氧化条件和分子机制,而且高水平表达时常容易聚集形成包涵体,限制了其使用,改善这一缺点的重要方法是通过信号肽实现蛋白质的分泌表达.信号肽一般存在于分泌蛋白的氨基端,能够引导蛋白质通过大肠杆菌中的Sec或/和Tat系统分泌至周质空间....     

Inefficient mismatch repair: genetic defects and down regulation

The mismatch repair system is involved in the maintenance of genomic integrity by editing DNA replication and recombination. However, although most mutations are neutral or deleterious, a mutator phenotype due to an inefficient mismatch repair may generate advantageous variants and may therefore be selected for. We review the evidence for inefficient mismatch repair due either to genetic defects in mismatch repair genes or to physiological conditions. Among natural isolates ofEscherichia coli andSalmonella enterica, about 1% are mutator bacteria, mostly deficient in mismatch repair (most of them defective in themutS gene). Characterization of mutators derived from laboratory strains led also to the isolation of mismatch repair mutants in which the most frequently found defects are inmutL andmutS. The correlation of the size of the antimutator genes with the frequency of their defective alleles amongE. coli andSalmonella strains reveals thatmutU mutants are underrepresented. Analysis of the progeny of a defined M13 phage heteroduplex DNA transfected intoE. coli cells shows that mismatch repair efficiency progressively decreases from the end of the exponential growth in K-12 and is variable among natural isolates. Implications of this defective mismatch repair activity for evolution and tumorigenesis will be discussed.     

Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches

Summary Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage , one of which has a 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.     

Frameshift mutagenesis of lambda prophage by 9-aminoacridine, proflavin and ICR-191

Summary The changes in DNA base sequence induced in the lambda cI gene in an E. coli lysogen have been determined following mutagenesis by three acridine derivatives: 9-aminoacridine and proflavin, which bind reversibly to DNA; and ICR-191, which attaches covalently to DNA through a half-mustard group. For all three derivatives, most mutations are +1 and-1 frameshifts in runs of adjacent G:C pairs. The specificity of mutagenesis at various sites is similar for all three compounds. Prophage in mutL host cells, deficient in mismatch repair, are much more susceptible to mutagenesis by 9-aminoacridine. The induced mutations are also frameshifts, and the site specificity is the same as in lysogens of wild type cells. Thus, additions or deletions of single bases can be corrected by the mismatch repair system, but mismatch repair does not play an important role in determining the sequence specificity of the mutational events.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

Mismatch repair in mammalian cells

A vital process in maintaining a low genetic error rate is the removal of mismatched bases in DNA. The importance of this process in E. coli is demonstrated by the 100–1000 fold increase in mutation frequency observed in cells deficient in this repair system⁽¹⁾. Mismatches can arise as a consequence of recombination, errors in replication and as a result of spontaneous chemical deamination, the latter process resulting in an estimated twelve T:G mismatches per genome per day in mammalian cells⁽²⁾. Recent studies, discussed here, provide evidence for the existence of specific mismatch repair systems in mammalian and human cells.     

Recombination,mutation and the origin of species

A major barrier to recombination between bacterial species lies in the mismatch repair system, a complex of proteins that has evolved to proof-read freshly replicated DNA. It now appears that a second system, involving an inducible DNA recombination, repair and mutagenesis pathway, also regulates interspecies recombination, but in a positive way, being required for recombination between Escherichia coli and Salmonella typhimurium⁽¹⁾. Thus the rate at which newly emerging species of bacteria diverge can be seen as a balance between a permissive state associated with inducible repair and recombination, and the proof-reading of intermediates in the recombination pathway by the mismatch correction system.     

Sequence-specific and DNA structure-dependent interactions of Escherichia coli MutS and human p53 with DNA

Many proteins involved in DNA repair systems interact with DNA that has structure altered from the typical B-form helix. Using magnetic beads to immobilize DNAs containing various types of structures, we evaluated the in vitro binding activities of two well-characterized DNA repair proteins, Escherichia coli MutS and human p53. E. coli MutS bound to double-stranded DNAs, with higher affinity for a G/T mismatch compared to a G/A mismatch and highest affinity for larger non-B-DNA structures. E. coli MutS bound best to DNA between pH 6 and 9. Experiments discriminated between modes of p53–DNA binding, and increasing ionic strength reduced p53 binding to nonspecific double-stranded DNA, but had minor effects on binding to consensus response sequences or single-stranded DNA. Compared to nonspecific DNA sequences, p53 bound with a higher affinity to mismatches and base insertions, while binding to various hairpin structures was similar to that observed to its consensus DNA sequence. For hairpins containing CTG repeats, the extent of p53 binding was proportional to the size of the repeat. In summary, using the flexibility of the magnetic bead separation assay we demonstrate that pH and ionic strength influence the binding of two DNA repair proteins to a variety of DNA structures.     

Endonuclease activities of MutLα and its homologs in DNA mismatch repair

MutLα is a key component of the DNA mismatch repair system in eukaryotes. The DNA mismatch repair system has several genetic stabilization functions. Of these functions, DNA mismatch repair is the major one. The loss of MutLα abolishes DNA mismatch repair, thereby predisposing humans to cancer. MutLα has an endonuclease activity that is required for DNA mismatch repair. The endonuclease activity of MutLα depends on the DQHA(X)₂E(X)₄E motif which is a part of the active site of the nuclease. This motif is also present in many bacterial MutL and eukaryotic MutLγ proteins, DNA mismatch repair system factors that are homologous to MutLα. Recent studies have shown that yeast MutLγ and several MutL proteins containing the DQHA(X)₂E(X)₄E motif possess endonuclease activities. Here, we review the endonuclease activities of MutLα and its homologs in the context of DNA mismatch repair.