Analysis of mutagenesis induced by a thermolabile T4 phage deoxycytidylate hydroxymethylase suggests localized deoxyribonucleotide pool imbalance

Summary To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34° C). One such marker, rII SN103, which showed the highest stimulation at 34° C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34° C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34° C cultures showed no significant deviations from values obtained at 30° C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the imitator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.     

Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro

Deoxyguanosine triphosphate is underrepresented among the four common deoxyribonucleoside triphosphates (dNTPs), typically accounting for just 5-10% of the total dNTP pool. We have asked whether this pool asymmetry affects the fidelity of DNA replication, by use of an in vitro assay in which an M13 phagemid containing the Escherichia coli lacZalpha gene and an SV40 replication origin is replicated by extracts of human cells. By monitoring reversion of either a TGA or TAA codon within the lacZalpha gene, we found that replication in "biologically biased" dNTPs, representing our estimate of the concentrations in HeLa cell nuclei, is not significantly more accurate than when measured in reaction mixtures containing the four dNTPs at equimolar concentrations. However, sequence analysis of revertants revealed significantly different patterns of mispairing events leading to mutation. During replication at biased dNTP levels, mutations at the site 5' to C in the template strand for the TGA triplet were less frequent than seen in equimolar reaction mixtures, suggesting that extension from mismatches at this site is relatively slow, and proofreading efficiency high, when dGTP is the next nucleotide to be incorporated. Mismatches opposite template C, which might have been favored by the low physiological concentrations of dGTP, were not favored in our in vitro system, although one particular substitution at this site, TGA-->TTA, was strongly favored at low [dGTP]. An excess of one dNTP was found in our system to be more mutagenic than a corresponding deficiency. We also estimated dNTP concentrations in non-transformed human fibroblasts and found that in vitro replication at these levels caused significantly fewer mutations than we observed under equimolar conditions (100 microM each dNTP). This increased replication fidelity may result from increased proofreading efficiency at the lower dNTP levels; however, replication rates were decreased only slightly at these non-transformed fibroblast concentrations.     

植物乳杆菌荚膜缺陷型菌株的诱变选育

以自主分离和鉴定的产荚膜多糖植物乳杆菌C88为出发菌株,采用亚硝基胍诱变、墨汁负染和显微镜观察筛选获得一株荚膜缺陷型突变株,命名为植物乳杆菌C88M3,经多次传代突变菌株具有良好的遗传稳定性。通过16SrDNA序列分析、菌株生长曲线和RAPD分析比较了野生型菌株和荚膜缺陷型菌株在遗传特性和产荚膜情况方面的差异。通过化学诱变方法获得了乳杆菌荚膜缺陷型菌株,对进一步研究荚膜多糖在乳杆菌益生性中的功能和作用机制具有重要意义。     

The hidden side of unstable DNA repeats: Mutagenesis at a distance

Structure-prone DNA repeats are common components of genomic DNA in all kingdoms of life. In humans, these repeats are linked to genomic instabilities that result in various hereditary disorders, including many cancers. It has long been known that DNA repeats are not only highly polymorphic in length but can also cause chromosomal fragility and stimulate gross chromosomal rearrangements, i.e., deletions, duplications, inversions, translocations and more complex shuffles. More recently, it has become clear that inherently unstable DNA repeats dramatically elevate mutation rates in surrounding DNA segments and that these mutations can occur up to ten kilobases away from the repetitive tract, a phenomenon we call repeat-induced mutagenesis (RIM). This review describes experimental data that led to the discovery and characterization of RIM and discusses the molecular mechanisms that could account for this phenomenon.     

头孢菌素C产生菌的诱变育种及培养基优化

通过对顶头孢霉（Cephalosporium acremonium）FC-01进行诱变选育及特定种子培养基的优化,提高了头孢菌素C的发酵产量。分别采用紫外-氯化锂和钴-60（60Co）γ射线对FC-01进行诱变选育,筛选到高产菌株FC-1-4和FC-4-2,产量较出发菌株分别提高了26%和54.5%。运用Plackett-Burman设计方法和响应面法对种子培养基进行优化,头孢菌素C发酵效价较对照分别提高了34.7%和13.2%,优化后的种子培养基主要成分为玉米浆3.70%、葡萄糖2.62%和硫酸镁0.15%,得到的菌株及相应的种子培养条件已成功应用在160M³工业发酵罐生产中,具有重要的工业生产能力。     

Replication stress triggered by nucleotide pool imbalance drives DNA damage and cGAS-STING pathway activation in NAFLD

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Mutagenesis after exposure of bacteria to ultraviolet light and delayed photoreversal

Summary The induction of mutations by ultraviolet light and delayed photoreversal in bacteria defective for SOS mutagenesis is discussed in terms of two models: the two-step misincorporation and bypass model, and the model involving simple deamination of cytosine-containing dimers. In phage S13 the latter appears to be the predominant mechanism. In Escherichia coli there is little evidence that the simple deamination mechanism is of any significance except in ung strains lacking uracil glycosylase where uracils left after photoreversal are not removed. Deamination might, however, occur during the operation of translesion synthesis via the two-step model and if it did, subsequent photoreversal would lead to the mutation being extended from one to both strands by uracil glycosylase repair rather than being removed.     

温莪术内生真菌Gibberella moniliformis EZG0807诱变及其诱变株遗传稳定性研究

利用大梯度超导磁体(JMT-16T50F)模拟失重和超重环境对温莪术内生真菌Gibberella moniliformis EZG0807进行诱变,以期得到代谢产物活性高、遗传稳定性好的菌株。诱变24 h、48 h和72 h后,通过稀释涂布平板法得到139株诱变菌株;经滤纸片抑菌法初筛和MTT法抗肿瘤细胞活性实验复筛,筛选出高活性诱变菌株M7226。采用群体传代的方法考察菌株M7226十代以内菌株的生长状况和次级代谢产物抗菌抗肿瘤活性的能力。结果显示活性内生真菌EZG0807经大梯度超导磁体诱变,筛选得到一株代谢产物活性高、遗传稳定性好的诱变菌株M7226,为后续次级代谢产物的分离纯化奠定基础,同时此法为真菌诱变育种提供了一种新的可供选择的方法。     

酵母过氧化物体生物合成缺陷突变株的诱变、筛选和鉴定

过氧化物体对生物的生长和发育非常重要,人类很多疾病就是由于过氧化物体生物合成缺陷引起。以解脂耶氏酵母E122为出发菌,采用硫酸二乙酯诱变,获得了两株过氧化物体生物合成缺陷突变株,其中一株为温度敏感的突变株。在正常生长条件下,突变株的免疫荧光分析显示弥散的染色模式,且在电镜下观察不到过氧化物体的形态结构。将克隆于表达载体pINA445上的目前所发现的与过氧化物体生物合成有关的基因转化这两株突变株,发现它们均不能恢复其在含油酸的培养基上的生长,表明这两个突变株是由与过氧化物体生物合成相关的新基因的突变引起。这两个突变株的获得为参与过氧化物体生物合成的新基因的发现奠定了基础。     

New structural and mechanistic insight into the A-rule and the instructional and non-instructional behavior of DNA photoproducts and other lesions

The A-rule in mutagenesis was originally proposed to explain the preponderance of X-->T mutations observed for abasic sites and UV damaged sites. It was deduced that when a polymerase was faced with a non-instructional lesion, typified by an abasic site, it would preferentially incorporate an A. In the absence of any other compelling explanation, any lesion causing an X-->T mutation has often been classified as non-instructional to account for its apparent lack of instructional ability. The A-rule and the classification of lesions as non-instructional were formulated before the active sites of any polymerases or the mechanism by which they synthesized DNA were known. Since then, much structural and kinetic data on DNA polymerases has emerged to suggest mechanistic explanations for the A-rule and the instructive and non-instructive behavior of lesions such as cis-syn dimers. Polymerases involved in the replication of undamaged DNA have highly constrained active sites that evolved to only accommodate the templating base and the complementary nucleotide and as a result are relatively intolerant of modifications that alter the size and shape of the nascent base pair. On the other hand, DNA damage bypass polymerases have much more open and less constrained active sites, which are much more tolerant of modifications. An otherwise instructional lesion would become non-instructional if it were unable to fit into the active site, and thereby behave transiently like an abasic site, leading to the insertion of whichever nucleotide is favored by the polymerase, generally an A. In this review, what is known about the active sites and mechanisms of replicative and DNA damage bypass polymerases will be discussed with regard to the A-rule and non-instructive behavior of lesions, typified by dipyrimidine photoproducts.     

鸡Mx蛋白基因诱变修饰及抗病活性

[目的]进一步研究鸡Mx蛋白第631位氨基酸的变异与鸡群抗病性的相关性.[方法]本实验利用PCR突变技术将鸡Mx蛋白基因的全长cDNA第2032位的碱基由G突变为A(既631位氨基酸的改变),并将突变的Mx基因插入真核表达载体pcDNA3.0,重组表达载体转染COS-Ⅰ细胞后,进行RT-PCR与间接免疫荧光(IFA)鉴定.[结果]对鸡Mx蛋白基因的cDNA进行PCR诱变修饰正确,构建了能够正确表达鸡Mx蛋白的重组真核表达载体;诱变修饰重组Mx蛋白对抗新城疫病毒(NDV)感染分析结果显示,重组Mx蛋白具有较强的抗新城疫病毒生物活性.[结论]为下一步研究鸡Mx蛋白的抗病机理与制备抗病转基因鸡奠定了坚实的基础.     

人U_1和U_2snRNA基因定位缺失和取代突变

 用寡聚核苷酸诱导的定位突变法,将人U_1和U_2snRNA基因的5'-端调控区域的一段能与SV_(40)T抗原相结合的DNA删去,造成缺失突变,改变这段DNA核苷酸的排列顺序,造成取代突变。突变株用原位杂交法筛选,由限制性内切酶电泳图谱分析和DNA顺序测定得到证实。突变率约为5％。     

The Discovery of Error-prone DNA Polymerase V and Its Unique Regulation by RecA and ATP

My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.     

应用聚合酶链反应突变和克隆HIV-lgag基因片段

作者设计并合成了一对突变引物PGO1和PGO2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、BamHI和ATG及TAA序列,以便于HlV-1gag基因序列的定向克隆和表达。用PCO1和PGO2作引物,采用PCR方法从HIV-1基因组DNA中扩增出一个长504bp的DNA片段,用EcoRl和BamHI双酶切位点将此片段定向克隆入pUC19质粒。将克隆基因插入M13mp18进行DNA序列分析,结果表明,该基因序列及读框完全正确,且在其5′末端突变出EcoRI位点和ATG起始码,3′末端突变出TAA终业码和BamHI位点,从而为该基因的表达研究奠定了基础。     

氦氖激光、紫外复合诱变天麻素产生菌华根霉LN-A的原生质体

本文通过研究酶组合、酶浓度、酶作用时间和菌龄等因素对天麻素产生菌华根霉(Rhizopus chinensis) LN-A原生质体制备和再生的影响, 总结出了原生质体制备和再生的最佳条件: 选用对数生长期的菌株, 以蜗牛酶5 mg/mL + 纤维素酶5 mg/mL + 溶菌酶2 mg/mL 30°C保温处理2 h, 原生质体形成率达5.8×107, 原生质体再生率为5.7%。在此基础上首次利用He-Ne激光、紫外线复合诱变天麻素合成菌的原生质体, 当选用15 mW的He-Ne激光辐射原生质体20 min, 再用紫外辐照150 s时获得了转化率及天麻素得率都明显提高的突变株, 其天麻素得率比出发菌株提高20%以上。     

A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch

Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid.     

Proline Scanning Mutagenesis Reveals a Role for the Flap Endonuclease-1 Helical Cap in Substrate Unpairing

The prototypical 5′-nuclease, flap endonuclease-1 (FEN1), catalyzes the essential removal of single-stranded flaps during DNA replication and repair. FEN1 hydrolyzes a specific phosphodiester bond one nucleotide into double-stranded DNA. This specificity arises from double nucleotide unpairing that places the scissile phosphate diester on active site divalent metal ions. Also related to FEN1 specificity is the helical arch, through which 5′-flaps, but not continuous DNAs, can thread. The arch contains basic residues (Lys-93 and Arg-100 in human FEN1 (hFEN1)) that are conserved by all 5′-nucleases and a cap region only present in enzymes that process DNAs with 5′ termini. Proline mutations (L97P, L111P, L130P) were introduced into the hFEN1 helical arch. Each mutation was severely detrimental to reaction. However, all proteins were at least as stable as wild-type (WT) hFEN1 and bound substrate with comparable affinity. Moreover, all mutants produced complexes with 5′-biotinylated substrate that, when captured with streptavidin, were resistant to challenge with competitor DNA. Removal of both conserved basic residues (K93A/R100A) was no more detrimental to reaction than the single mutation R100A, but much less severe than L97P. The ability of protein-Ca²⁺ to rearrange 2-aminopurine-containing substrates was monitored by low energy CD. Although L97P and K93A/R100A retained the ability to unpair substrates, the cap mutants L111P and L130P did not. Taken together, these data challenge current assumptions related to 5′-nuclease family mechanism. Conserved basic amino acids are not required for double nucleotide unpairing and appear to act cooperatively, whereas the helical cap plays an unexpected role in hFEN1-substrate rearrangement.     

Charting the Signal Trajectory in a Light-Oxygen-Voltage Photoreceptor by Random Mutagenesis and Covariance Analysis

Modular signal receptors empower organisms to process environmental stimuli into adequate physiological responses. At the molecular level, a sensor module receives signals and processes the inherent information into changes of biological activity of an effector module. To better understand the molecular bases underpinning these processes, we analyzed signal reception and processing in the dimeric light-oxygen-voltage (LOV) blue light receptor YF1 that serves as a paradigm for the widespread Per-ARNT-Sim (PAS) signal receptors. Random mutagenesis identifies numerous YF1 variants in which biological activity is retained but where light regulation is abolished or inverted. One group of variants carries mutations within the LOV photosensor that disrupt proper coupling of the flavin-nucleotide chromophore to the protein scaffold. Another larger group bears mutations that cluster at the dyad interface and disrupt signal transmission to two coaxial coiled-coils that connect to the effector. Sequence covariation implies wide conservation of structural and mechanistic motifs, as also borne out by comparison to several PAS domains in which mutations leading to disruption of signal transduction consistently map to confined regions broadly equivalent to those identified in YF1. Not only do these data provide insight into general mechanisms of signal transduction, but also they establish concrete means for customized reprogramming of signal receptors.     

细菌感受态细胞摄取和分泌DNA的相关性研究I.含有质粒的枯草芽孢杆菌自然感受态缺陷突变株的诱变和分离

以携带ｐＵＢ１１０质粒的枯草芽孢杆菌ＢＲ１５１菌株为出发菌株,利用亚硝基胍作诱变剂,直接在ＬＢ平板上进行诱变。从２９４９个菌落中筛选出一个转化频率低于出发菌株２～３个数量级的突变株,并对其营养缺陷型、ＵＶ的敏感性及Ｋｍ抗性和质粒进行了检测,确证其转化能力降低为自然感受态缺陷而非营养缺陷型的改变或重组缺陷所致