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生化重要概念公司内部档案编码：[OPPTR-OPPT28-OPPTL98-OPPNN08]生化重要概念1.重要概念：primary structure of protein蛋白质的一级结构是指蛋白质多肽链中通过肽键连接起来的氨基酸的排列顺序，即多肽链的线状结构2.模体（motif ）：在蛋白质分子中，若干具有二级结构的肽段在空间上相互接近，形成具有特殊功能的结构区域3. 重要概念：allosteric effect由于蛋白质分子构象改变而导致蛋白质分子功能发生改变的现象称为变构效应4. 重要概念：denaturation of protein在某些物理或化学因素的作用下，蛋白质严格的空间结构被破坏（不包括肽键的断裂），从而引起蛋白质若干理化性质和生物学性质的改变，称为蛋白质的变性5. 重要概念：primary structure of DNADNA的一级结构就是指DNA分子中脱氧核糖核苷酸的排列顺序及连接方式（3,5-磷酸二酯键）6.重要概念：primary structure of RNARNA的一级结构就是指RNA分子中核糖核苷酸的排列顺序及连接方式。
7.重要概念：geneDNA分子中具有特定生物学功能的片段称为基因。
8.重要概念：genome一个生物体的全部DNA序列称为基因组。
9.重要概念：restriction endonuclease能识别特定的核苷酸顺序，并从特定位点水解核酸的内切酶称为限制性核酸内切酶（限制酶，restriction enzyme）10.重要概念：coenzyme & prosthetic group结合酶分子中与酶的催化活性有关的耐热小分子有机化合物称为辅酶。
与酶蛋白共价结合的辅酶称为辅基。
11. 重要概念：vitamin维生素是指一类维持细胞正常功能所必需的，但在生物体内不能自身合成而必须由食物供给的小分子有机化合物。
12重要概念：isoenzyme在同一种属中，催化活性相同而酶蛋白的分子结构，理化性质及免疫学性质不同的一组酶称为同工酶。
供学生使用的《生物化学》重要术语中英语对照碳水化合物〔carbohydrate〕单糖〔monosaccharide〕寡糖〔oligosaccharide〕多糖〔polysaccharide〕醛糖〔aldose〕酮糖〔ketose〕蔗糖〔sucrose〕乳糖〔lactose〕麦芽糖〔maltose〕纤维二糖〔cellobiose〕多糖〔polysaccharides〕淀粉〔starch〕直链淀粉〔amylose〕支链淀粉〔amylopectin〕纤维素〔cellulose〕半纤维素〔hemicellulose〕糖原〔glycogen〕几丁质〔chitin〕糖胺聚糖〔glycosaminolgycan〕脂类〔lipids〕脂肪酸〔fatty acid〕甘油三酯〔glycerol triester〕亲水脂类〔amphipathic lipids〕蜡〔wax〕磷酸甘油脂〔phosphoglyceride〕甘油磷脂〔glycerophospholipid〕磷脂酰胆碱〔phosphatidylcholine〕磷脂酰乙醇胺〔phosphatidylethanolamine〕磷脂酰丝氨酸〔phoshatidylserine〕磷脂酰肌醇〔phosphatidylinositol, PI〕肌醇三磷酸〔inositol-1,4,5-trisphosphate，IP3〕二脂酰甘油〔diacylglycerol，DAG〕磷脂酸〔phosphatidic acid，PA〕磷脂酶A2〔phospholipase A2，PLA2〕磷脂酶C〔phospholipase C，PLC〕磷脂酶D〔phospholipase D，PLD〕溶血磷脂(1ysophospholipid)鞘磷脂〔sphingomyelin〕神经酰胺〔ceramide〕类固醇〔steroids〕萜类〔terpenes〕胆固醇〔cholesterol〕麦角固醇〔ergosterol〕蛋白质 protein简单蛋白质 simple protein氨基酸 amino acid结合蛋白质 conjugated protein多肽 polypeptide肽 peptide肽键 peptide bond介电常数 dielectric constant德华力 van der waals force层析法 chromatography吸附层析法 adsorption chromatography分配系数 partition or distribution confficient活性肽 active peptide二硫键 disulfide bond兼性离子 zwitterion一级结构 primary structure疏水效应 hydrophobic effectSDS-聚丙烯酰胺凝胶电泳 SDS-PAGE毛细管电泳〔capillary eletrophoresis, CE〕离子交换层析 ion exchange chromatography 同源蛋白 homologous protein构象 conformation构象角 conformatiomal angle糖脂〔glycolipid〕糖基甘油酯〔glycosylglyceride〕鞘糖脂〔glycosphingolipid〕脑苷脂〔cerebroside〕N-乙酰神经氨酸〔N-acetylneuraminic acid〕神经节苷脂〔ganglioside〕硫酸脑苷脂〔cerebroside sulfate〕糖蛋白〔glycoproteins〕蛋白聚糖〔proteoglycans〕生物膜〔biomembrane〕膜脂〔membrane lipids〕膜蛋白〔membrane proteins〕脂质双层分子〔lipid bilayers〕外周蛋白〔peripheral protein〕外源性〔extrinsic protein〕在蛋白〔integral protein〕源性蛋白〔intrinsic protein〕跨膜蛋白〔transmembrane proteins〕流动镶嵌模型〔fluid mosaic model〕简单扩散〔simple diffusion〕协助扩散〔facilitated diffusion〕被动运输〔passive transport〕主动运输〔active transport〕介导性运输〔mediated transport〕非介导性运输〔nonmediated transport〕载体蛋白〔carrier protein〕通道蛋白〔channel protein〕离子通道〔ionic channel〕离子载体〔ionophore〕吞作用〔endocytosis〕胞饮作用〞〔pinocytosis〕外排作用〔exocytosis〕基团转移〔group translocation〕脂蛋白〔lipoprotein〕染色体〔chromosome〕染色质〔chromatin〕组蛋白〔histone〕核小体〔nucleosome〕病毒〔virus〕噬菌体〔bacteriophage或简称phage〕变性 denaturation沉降系数〔S〕Svedberg(S)抗体 antibody亲和层析法 affinity chromatography盐溶 salting in盐析 salting out二级结构 secondary structure三级结构 tertiary structure -螺旋-helix超二级结构 super-secondaery structure 结构域 structure domain氢键 hydrogen bend疏水相互作用 hydrophoblic interaction 肌红蛋白 myoglobin寡聚蛋白质 oligomeric protein无规那么卷曲 randon coil复性 renaturation镰刀状细胞贫血病 sickle-cell anermia酶〔enzyme〕酶的专一性〔specificity〕单体酶〔monomeric enzyme〕寡聚酶〔oligomeric enzyme〕多酶复合体系〔multienzyme system〕酶活性中心〔active center of enzyme〕催化基团〔catalytic site〕酶原〔zymogen or proenzyme〕诱导契合〔induced-fit theory〕抗体酶〔abzyme〕酸碱催化〔acid-base catalysis〕共价催化(covalent catalysis)激活剂〔activator〕抑制剂〔inhibitor〕可逆抑制〔reversible inhibition〕竞争性抑制作用〔competitive inhibition〕非竞争性抑制作用〔noncompetitive inhibition〕调节酶〔modulator〕别构酶〔allosteric enzyme〕同配位效应〔isosteric effect〕变构效应〔allosteric effect〕变构激活〔allosteric activation 〕正协同效应〔positive cooperative effect〕负协同效应〔negative cooperative effect〕效应物〔effector〕维生素〔vitamin〕维生素缺少症〔avitaminosis〕调节中心〔regulatory center〕催化亚基〔catalytic subunit〕调节亚基〔regulatory subunit〕诱导酶〔induced enzyme〕结构酶〔structural enzyme〕核酶〔ribozyme〕辅酶〔coenzyme〕比活力〔specific activity〕脱氧核酶〔deoxyribozyme〕酶工程〔enzyme engineering〕酶纯度〔purity of enzyme〕酶活力〔enzyme activity〕-淀粉酶〔-amylase〕-淀粉酶〔-amylase〕脱支酶〔debranching enzyme〕淀粉的磷酸化酶〔amylophosphorylase〕糖酵解〔glycolysis〕三羧酸循环〔tricarboxylic acid cycle，TCA〕磷酸戊糖途径〔pentose phosphatepathway，PPP〕生物氧化〔biological oxidation〕烟酰胺脱氢酶类〔nicotinamide dehydrogenase〕黄素脱氢酶类〔flavin dehydrogenase〕铁硫蛋白类〔iron-sulfur protein〕泛醌〔ubiquinone〕细胞色素类〔cytochromes〕细胞色素氧化酶〔cytochromeoxidase〕鱼藤酮〔rotenone〕安密妥〔amytal〕杀粉蝶菌素〔piericidine〕抗霉素A〔antimycin A〕底物水平磷酸化〔substrate-level phosphorylation〕氧化磷酸化〔oxidative phosphorylation〕化学渗透假说〔chemiosmotic coupling hypothesis〕化学偶联假说〔chemical coupling hypothesis〕构象偶联假说〔conformational coupling hypothesis〕甘油-磷酸穿梭途径〔glycerophosphate shuttle〕苹果酸-天冬氨酸穿梭途径〔malate- aspartate shuttle〕异柠檬酸穿梭途径〔isocitrate shuttle〕能荷〔energy charge〕肉碱〔肉毒碱，carnitine〕乙醛酸体〔乙醛酸循环体，glyoxysome〕乙醛酸循环〔glyoxylate cycle〕酮体〔ketone bodies〕饱和脂肪酸的从头合成〔de novo synthesis〕谷氨酸脱氢酶〔glutamate dehydrogenase, GDH〕转氨基作用〔transamination〕转氨酶〔transaminase〕磷酸吡哆醛〔pyridoxal phosphate，PLP〕谷丙转氨酶〔glutamic pyruvic transaminase，GPT或 alanine transaminase，ALT〕谷草转氨酶〔glutamic oxaloacetic transaminase，GOT或 aspartate transaminase，AST〕γ-谷氨酰-半胱氨酸合成酶〔γ-glutamyl systeine synthetase，γ-ECS〕谷胱甘肽〔glutathione〕谷胱甘肽合成酶〔glutathione synthetase〕生物固氮〔biological nitrogen fixation〕固氮酶〔nitrogenase〕自身固氮微生物〔diazatrophs〕共生固氮微生物〔symbiotic microorganism〕硝酸复原酶〔nitrate reductase，NR〕亚硝酸复原酶〔nitrite reductase，NiR〕谷氨酸合酶〔glutamate: oxo-glutarate aminotransferase，GOGAT〕谷氨酰胺合成酶〔glutamine synthetase，GS〕腺苷-5'-磷酸硫酸酐〔adenosine-5'-phosphosulfate，APS〕3'-磷酸腺酐-5'-磷酰硫酸〔3'-phosphoadenosine-5'-phosphosulfate，PAPS〕5-磷酸核糖焦磷酸〔phosphoribosyl pyrophosphaet，PRPP〕天冬氨酸转氨甲酰酶〔aspartate trsnscarbamoy lase〕腺嘌呤磷酸核糖转移酶〔adenine phosphoribosyl fransferase，APRT〕黄嘌呤-鸟嘌呤磷酸核糖转移酶〔hypoxanthineguanine phosphoribosyl transferase，HGPRT〕谷胱甘肽复原酶〔glutathione reductase，GR〕谷氧还蛋白〔glutaredoxin〕谷氧还蛋白复原酶〔glutaredoxin reductase〕胸腺嘧啶核苷酸合酶〔thymidylate synthase〕DNA复制〔DNA replication〕中心法那么〔central dogma〕冈崎片段〔Okazaki fragement〕前导链〔leading strand〕滞后链〔lagging strand〕引物〔primer〕复制叉〔replication fork〕半保存式复制〔semiconservative replication〕模板〔template〕反转录〔reverse transcription〕转换〔transition〕颠换〔transversion〕错配修复〔mismatch repair〕核苷酸切除修复〔nucleotide excision repair〕碱基切除修复〔base excision repair〕同源重组〔homologous recombination〕特异性重组〔site-specific recombination〕转座子〔transposon〕启动子〔promoter〕限制性切酶〔restriction endonuclease 〕修饰〔modification〕单链结合蛋白〔single stranded binding proteins,SSB〕遗传密码〔genetic code〕读码框架〔reading frame〕移码突变〔frame-shift mutation〕简并性〔degeneracy〕同义密码子〔synonymous codon〕起始密码子〔initiatlon codon〕终止密码子〔termination codon〕摆动假说〔wobble hypothesis)同功受体tRNA〔isoaccepting tRNA〕反密码子〔anticodon〕多核糖体〔polyribisome〕氨酰-tRNA合成酶〔aminoacyl-tRNA synthetase〕Shine –Dalgarno序列〔Shine –Dalgarno sequence〕起始因子〔initiation factor〕延伸因子〔elongation factor〕释放因子〔release factor〕转肽〔transpeptidation〕移位〔translocation〕分子伴侣〔molecular chapeones〕共翻译转移〔co-translational translocation〕翻译后转移〔post-translational translocation〕信号肽〔signal sequence〕信号识别颗粒〔signal recognition particle SPR〕代〔metabolism〕代调节〔metabolic regulation〕共价修饰〔covalent modification〕反应抑制〔feedback inhibition〕操纵子模型〔operon model〕衰减作用〔attenuation〕级联放大作用〔amplification cascade〕变〔别〕构效应〔allosteric effect〕诱导和阻遏〔induction and repression〕蛋白激酶 C 〔protein kinase C，PKC〕第二信使〔second messenger〕受体〔receptor〕G 蛋白〔guanosine triphosphate-bindingprotein〕信号转导〔signal transduction〕钙调素〔calmodulin，CaM〕磷酯酶〔phospholipase C，PLC〕。
Botany 植物学Cell theory细胞学说cell membrane细胞膜nucleus细胞核Organelle细胞器cell wall细胞壁cytoplasm细胞质protoplast原生质体cell cycle细胞周期mitochondrion线粒体photosynthesis 光合作用unit membrane单位膜chloroplast叶绿体chlorophyll叶绿素xanthophyll 叶黄素carotene胡萝卜素golgiosome高尔基体ribosome核糖体lysosome溶酶体microfilament 微丝nuclear fission核分裂reproduction生殖primary wall初生壁secondary wall次生壁plasmodesma胞间连丝mitosis 有丝分裂amitosis无丝分裂meiosis减数分裂cytokinesis胞质分裂interphase间期prophase先期metaphase中期anaphase后期telophase末期tissue 组织pistil雌蕊stamen 雄蕊ovary 子房pollination传粉pollen tube花粉管porogamy珠孔受精chalazogamy合点受精mesogamy中部受精apomixis无交融生殖apogamy无配子生殖patrogenesis孤雄生殖parthenogensis 孤雌生殖apospory无孢子生殖pericarp果皮life history生活史root system根系main root主根lateral root侧根taproot system直根系fibrous root system须根系cortex 皮层vascular cylinder维管柱pericycle 中柱鞘xylem ray木射线vascular ray维管射线phloem ray韧皮射线root cap根冠Casparian strip凯氏带primary xylem初生木质部primary phloem 初生韧皮部vascular ray维管射线xylem ray木射线phelloderm栓内层phloem ray韧皮射线embryo胚homologous organ同源器官analogous organ 同功器官endosperm胚乳seed coat种皮radicle 胚根plumule 胚芽hypocotyl下胚轴cotyledon子叶dormancy 休眠seed germination种子萌生eukaryote真核生物prokaryote原核生物algae 藻类blue-green algae 蓝藻trichogyne受精丝精选文库mucopolysaccharide 黏多糖gelatinous sheath 胶质鞘exospore外生孢子heterosexual cell异性细胞green algae绿藻isogamy同配生殖anisogamy 异配生殖anisogamy卵式生殖zygogamy接合生殖haploid 单倍体diploid 二倍体polyploid多倍体carposporophyte孢子体brown algae褐藻sea-tangle海带agar 琼脂fungi 菌类parasitism寄生saprophytic腐生的lichen 地衣archegonium颈卵器antheridium精子器antiphyte孢子体gametophyte配子体protonema原丝体bryophyta苔藓植物cruciferae十字花科vascular plants 微管植物aquatic plant 水生植物salicaceae杨柳科angiosperm被子植物endoplasmic reticulum内质网vegetative reproduction营养生殖intercellular layer 胞间层phellogen& cork cambium木栓形成层asexual reproduction无性生殖sexual propagation有性生殖tetradynamous stamen四强雄蕊didynamous stamen二强雄蕊monodelphous stamen单体雄蕊diadelphous stamen二体雄蕊triadelphous stamen三体雄蕊polyadelphous stamens多体雄蕊synantherous stamen聚药雄蕊primary wall cells初生壁细胞vegetative cell营养细胞male sterility雄性不育filiform apparatus 丝状器meristem zone分生区elongation zone伸长区maturation zone成熟区embryophyte 有胚植物specific parasitism专性寄生specific saprophyte 专性腐生facultative parasitism兼性寄生facultative saprophyte兼性腐生sexual generation有性世代asexual generation 无性世代Zoology 动物学cell 细胞prokaryotic cell原核细胞eukaryotic cell真核细胞protein 蛋白质nucleic acid核酸carbohydrate糖lipid 脂质protoplasm原生质inclusion内含物cell cycle细胞周期pulmonary alveolus肺泡flagellum鞭毛food vacuole食品泡pinocytosis胞饮作用fission裂体生殖microgamete小配子zygote 合子microtubule微管contraction silk缩短丝merogenesis卵裂blastocoele囊胚腔complete cleavage 完整卵裂layering分层cynapse突触myoneme肌丝myocyte肌细胞mesoglea 中胶层monoecism雌雄同体dioecism雌雄异体velum 缘膜radial symmetry辐射对称nerve net神经网planula浮浪幼虫bilateral symmetry 双侧对称mesoderm中胚层tubule cell管细胞osmoregulation浸透调理acetabulum腹吸盘oral sucker口吸盘metacercaria囊蚴pseudocoel假体腔cuticle 角质膜cloacal pore泄殖孔renette腺肾细emunctory排泄管resting egg休眠卵metamere体节metamerism分节现象sense organ感觉器periostracum壳皮层prismatic layer壳层nacreous layer珍珠层veliger面盘幼虫glochidium钩介幼虫adductor闭壳肌segmentation异律分节linear animal线形动物pericardial cavity围心腔cervical vertebra颈椎sacral vertebra荐椎pulmonary vein肺静脉precaval vein前腔静脉bladder气囊middle ear 中耳tympanum cavity中耳腔amnion羊膜neopallium新皮层lagena瓶状囊wishbone 叉骨postcaval vein 后腔静脉glandular stomach腺胃air sac气囊salt gland盐腺sclerotic ring巩膜骨viviparity 胎生placenta胎盘allantois尿囊rumen 瘤胃bursa of fabricius腔上囊masticatory stomach 肌胃reticulum网胃omasum瓣胃abomasum皱胃cochlea耳蜗earthworm 蚯蚓internal naris内鼻孔amniota羊膜动物arthropod节肢动物coelenterate 腔肠动物annelid环节动物cell membrane&plasma membrane细胞膜epithelial tissue上皮组织connective tissue结缔组织cartilage tissue软骨组织osseous tissue骨组织muscular tissue肌肉组织cardiac muscle心肌intercalated disc闰盘Nissl's body 尼氏小体colony &group集体meroblastic cleavage不完整卵裂colonial theory集体说gastrovascular cavity消化循环腔muscle system肌肉系统excretory system排泄系统reproductive system 生殖系统digestive system消化系统archinephric duct原肾管basal lamina & basal membrane基膜cross-fertilization异体受精self-fertilization自体受精final host 终寄主first intermidate host第一中间寄主semicircular canal半规管second intermediate host 第二中间寄主Genetics遗传学heredity遗传variation 变异gene 基因pisum sativum豌豆segregation 分别gamete生殖细胞zygote合子allele等位基因genotype基因型phenotype 表现型test cross测交oryza sativa水稻diploid二倍体haploid单倍体centromere着丝粒satellite随体linker 连丝mitosis 有丝分裂mesoblast 中胚层spindle纺锤体interphase 间期spindle fiber纺锤丝vicia faba蚕豆nucleoplasm核质spermatogenous精原细胞oogonium卵原细胞spermatid精美胞Phenocopy拟表型epistasis上位效应mutant突变型gametic lethal配子致死zygotic lethal合子致死autosome 常染色体dominant lethal显性致死carrier携带者homozygote纯合体heterozygote杂合体genotype基因型phenotype表现型linkage group连锁群interference干预coincidence 并发率genetic map遗传学图wild type 野生型mutation突变heterokaryon异核体auxotroph营养缺点型strain菌株recipient受体donor 供体fragment 片段induction引诱prophage原噬菌体transduction 转导Mendel ’ s laws 孟德尔定律law of segregation分别定律first filial generation子一代parental generation亲代dominant character显性性状recessive character隐性性状hereditary determinant遗传因子parental combination亲组合recombination重组合punnett square 棋盘法Mendelian character孟德尔性状primary constriction初级缢痕secondary constriction次级缢痕nucleolar organizer核仁形成区first polar body第一极体second polar body第二极体sister chromatids姐妹染色单体female gametic nucleus卵核multiple alleles复等位基因sex-chromosome性染色体sex-linked inheritance伴性遗传primary constriction初级缢痕secondary constriction次级缢痕complementary gene互补基因homologous chromosome同源染色体sister chromatids姐妹染色单体secondary oocyte次级卵母细胞three-point testcross三点测交primary spermatocyte初级精母细胞secondary spermatocyte次级精母细胞first division segregation第一次分裂分别second division segregation第二次分裂分别law of independent assortment自由组合定律Biochemistry生物化学essential element必要元素trace elements微量元素proteoglycan蛋白聚糖amino acid氨基酸primary structure一级构造random coil无规卷曲structural domain构造域subunit 亚基degeneration变性adenine腺嘌呤guanine鸟嘌呤cytosine胞嘧啶thymine胸腺嘧啶uracil 尿嘧啶nucleoside核苷nucleotide核苷酸base pairing碱基配对base pair碱基对数base 碱基数gyrase 旋转酶nucleosome核小体complementary DNA互补 DNA plasmid质粒transposons转座子repetitive sequence重复序列exon 外显子intron 内含子variable loop 可变环ribonuclease核糖核酸酶renaturation复性hyperchromic effect添色效应base stacking force碱基聚积力annealing退火melting-out temperature 溶化温度hypochromic effect 减色效应maltose 麦芽糖sucrose蔗糖lactose 乳糖starch淀粉glycogen糖原cellulose 纤维素cellulase纤维素酶selectivity选择性substrate 底物holoenzyme全酶cofactor辅因子coenzyme 辅酶oxidase 氧化酶metabolism新陈代谢assimilation同化作用catabolism异化作用metabolite代谢产物biological oxidation生物氧化cytochrome细胞色素rotenone鱼藤酮amytal 阿密妥antimycin A抗霉素 A cyanide氰化物glycolysis 糖酵解ethanol乙醇citrate 柠檬酸cis-aconitate顺乌头酸succinic acid琥珀酸oxaloacetic acid草酰乙酸acetyl-coenzyme乙酰辅酶fumarate延胡索酸glyoxylate cycle乙醛酸循环malate 苹果酸fatty acid脂肪酸carbon unit一碳单位replicon 复制子core enzyme核心酶primosome引起体Okazaki fragment冈崎片段leading chain前导链lagging strand后随链terminator 停止子telomere 端粒telomerase端粒酶replication fork 复制叉vector 载体promoter启动子terminator 停止子operon操控子codon 密码子degeneracy简并性hormone 激素citric acid cycle柠檬酸循环deamination 脱氨基作用urea cycle尿素循环transfer RNA转移 RNA ribosome RNA核糖体 RNA metabolic regulation代谢调理feedback regulation反应调理structural gene构造基因promoter gene启动基因operator gene操控基因regulator gene调理基因termination factor 停止因子triplet code 三联体密码initiator codon开端密码termination codon停止密码semiconservative replication半保存复制ornithine cycle 鸟氨酸循环ketogenic amino acid生酮氨基酸glucogenic amino acid生糖氨基酸oxidative deamination氧化脱氨作用transamination转氨基作用reverse transcription逆转录decarboxylation脱羧作用semidiscontinuous replication半不连续复制reverse transcriptase逆转录酶missense mutation错义突变synonymous mutation同义突变neutral mutation中性突变nonsense mutation无义突变phosphatidic acid磷脂酸essential amino acids必要氨基酸dihydrouracil loop二氢尿嘧啶环anticodon loop反密码子环double-strand circular DNA双链环形 DNA superhelical DNA超螺旋 DNA open circular DNA开环 DNA linear DNA线形 DNAbase stacking force碱基聚积力secondary structure二级构造super-secondary structure超二级构造tertiary structure三级构造quaternary structure四级构造negative supercoil DNA负超螺旋 DNA positive supercoil DNA正超螺旋 DNAGlyceraldehyde-3-phosphate甘油醛-3-二磷酸glucogenic and ketogenic amino acid生糖兼生酮氨基酸restriction endonuclease限制性内切酶polymerase chain reaction聚合酶链反响精选文库Microbiology 微生物学living creatures生物culture medium 培育基lawn 菌苔culture plate培育平板bacteria细菌archaea古生菌eukaryote真核生物prokaryote原核生物protozoan原生动物hypha 菌丝mycoplasma支原体yeast 酵母菌plasmolysis质壁分别Escherichia Coli 大肠杆菌murein 胞壁质peptidoglycan肽聚糖mucopeptide 黏肽outer membrane 外膜chromosome染色体nucleolus核仁nucleoid拟核chromatin染色质centromere着丝粒telomere端粒protoplast原生质体mycoplasma支原体glycoprotein糖蛋白mesosome间体cytoplasm细胞质megnetosome磁小体nucleoid拟核glycocalyx糖被capsule荚膜flagellum鞭毛lysosome溶酶体chloroplast 叶绿体thylakoid类囊体inorganic salt 无机盐peptone蛋白胨sulfur bacteria硫细菌beef extract牛肉膏vitamin维生素inclusion body内含物lithotroph无机营养型medium培育基agar 琼脂organotroph有机营养型antiport 逆向运输active transport主动运输pinocytosis胞饮作用catabolism 分解代谢passive transport被动运输uniport 单向运输anabolism合成代谢fermentation发酵batch culture 分批培育log phase对数生长久stationary phase 稳固生长久lag phase迟延期decline phase衰灭期aerobe好氧菌antibiotic抗生素antigenome反基因组transformation转变genome基因组plasmid 质粒transforming factor转变因子diploid二倍体haploid单倍体transposable element转座因子conjugation接合作用transposon转座子phenotype 表型genotype 基因型auxotroph 营养缺点型wild-type野生型transition变换transversion颠换spontaneous mutation自觉突变reverse mutation答复突变sexduction性导精选文库recombination repair重组修复repressor 隔绝蛋白corepressor辅隔绝物clone 克隆denaturation变性annealing退火extension延长cloning vector克隆载体replicon复制子telomere端粒cohesive end黏性尾端promoter启动子terminator停止子gene therapy基因治疗phylogeny系统发育ammonification氨化作用nitrification 硝化作用denitrification反硝化作用expression vector表达载体aerobic respiration有氧呼吸anaerobic respiration无氧呼吸origin of replication复制开端点incompatibility不亲和性gene mutation基因突变synonymous mutation同义突变chromosomal aberration染色体畸变missense mutation错义突变frame-shift mutation移码突变lactose operon乳糖操控子negative transcription control负转录调控tryptophan operon色氨酸操控子cytoplasmic inheritance细胞质遗传genetic engineering基因工程recombinant DNA technology重组 DNA 技术palindromic structure回文构造spread plate method涂布平板法pour plate method倾注培育法streak plate method平板划线法shake tube method稀释摇管法continuous culture连续培育。
【生物化学：名词解释大全】第一章 蛋 白 质1．两性离子（dipolarion）2．必需氨基酸（essential amino acid）3．等电点(isoelectric point,pI)4．稀有氨基酸(rare amino acid)5．非蛋白质氨基酸(nonprotein amino acid)6．构型(configuration)7．蛋白质的一级结构(protein primary structure) 8．构象(conformation)9．蛋白质的二级结构(protein secondary structure) 10．结构域(domain)11．蛋白质的三级结构(protein tertiary structure) 12．氢键(hydrogen bond)13．蛋白质的四级结构(protein quaternary structure) 14．离子键(ionic bond)15．超二级结构(super-secondary structure)16．疏水键(hydrophobic bond)17．范德华力( van der Waals force)18．盐析(salting out)19．盐溶(salting in)20．蛋白质的变性(denaturation)21．蛋白质的复性(renaturation)22．蛋白质的沉淀作用(precipitation)23．凝胶电泳（gel electrophoresis）24．层析（chromatography）第二章 核 酸1．单核苷酸(mononucleotide)2．磷酸二酯键（phosphodiester bonds）3．不对称比率（dissymmetry ratio）4．碱基互补规律(complementary base pairing) 5．反密码子（anticodon）6．顺反子（cistron）7．核酸的变性与复性（denaturation、renaturation）8．退火（annealing）9．增色效应（hyper chromic effect）10．减色效应（hypo chromic effect）11．噬菌体（phage）12．发夹结构（hairpin structure）13．DNA 的熔解温度（melting temperature T m）14．分子杂交（molecular hybridization）15．环化核苷酸(cyclic nucleotide)第三章 酶与辅酶1．米氏常数（K m 值）2．底物专一性（substrate specificity）3．辅基（prosthetic group）4．单体酶（monomeric enzyme）5．寡聚酶（oligomeric enzyme）6．多酶体系（multienzyme system）7．激活剂（activator）8．抑制剂（inhibitor inhibiton）9．变构酶（allosteric enzyme）10．同工酶（isozyme）11．诱导酶（induced enzyme）12．酶原（zymogen）13．酶的比活力（enzymatic compare energy）14．活性中心（active center）第四章 生物氧化与氧化磷酸化1． 生物氧化（biological oxidation）2． 呼吸链（respiratory chain）3． 氧化磷酸化（oxidative phosphorylation）4． 磷氧比P/O（P/O）5． 底物水平磷酸化（substrate level phosphorylation）6． 能荷（energy charg第五章 糖 代 谢1．糖异生(glycogenolysis)2．Q 酶(Q-enzyme)3．乳酸循环(lactate cycle)4．发酵(fermentation)5．变构调节(allosteric regulation)6．糖酵解途径(glycolytic pathway)7．糖的有氧氧化(aerobic oxidation)8．肝糖原分解(glycogenolysis)9．磷酸戊糖途径(pentose phosphate pathway)10．D-酶（D-enzyme）11．糖核苷酸（sugar-nucleotide）第六章 脂类代谢1． 必需脂肪酸（essential fatty acid）2． 脂肪酸的α-氧化(α- oxidation)3． 脂肪酸的β-氧化(β- oxidation)4． 脂肪酸的ω-氧化(ω- oxidation)5． 乙醛酸循环（glyoxylate cycle）6． 柠檬酸穿梭(citriate shuttle)7． 乙酰CoA 羧化酶系（acetyl-CoA carnoxylase）8． 脂肪酸合成酶系统（fatty acid synthase system）第八章 含氮化合物代谢1．蛋白酶（Proteinase）2．肽酶（Peptidase）3．氮平衡（Nitrogen balance）4．生物固氮（Biological nitrogen fixation）5．硝酸还原作用（Nitrate reduction）6．氨的同化（Incorporation of ammonium ions into organic molecules）7．转氨作用（Transamination）8．尿素循环（Urea cycle）9．生糖氨基酸（Glucogenic amino acid）10．生酮氨基酸（Ketogenic amino acid）11．核酸酶（Nuclease）12．限制性核酸内切酶（Restriction endonuclease）13．氨基蝶呤（Aminopterin）14．一碳单位（One carbon unit）第九章 核酸的生物合成1．半保留复制（semiconservative replication）2．不对称转录（asymmetric trancription）3．逆转录（reverse transcription）4．冈崎片段（Okazaki fragment）5．复制叉（replication fork）6．领头链（leading strand）7．随后链（lagging strand）8．有意义链（sense strand）9．光复活（photoreactivation）10．重组修复（recombination repair）11．内含子（intron）12．外显子（exon）13．基因载体（genonic vector）14．质粒（plasmid）第十一章 代谢调节1．诱导酶（Inducible enzyme）2．标兵酶（Pacemaker enzyme）3．操纵子（Operon）4．衰减子（Attenuator）5．阻遏物（Repressor）6．辅阻遏物（Corepressor）7．降解物基因活化蛋白（Catabolic gene activator protein）8．腺苷酸环化酶（Adenylate cyclase）9．共价修饰（Covalent modification）10．级联系统（Cascade system）11．反馈抑制（Feedback inhibition）12．交叉调节（Cross regulation）13．前馈激活（Feedforward activation）14．钙调蛋白（Calmodulin）第十二章 蛋白质的生物合成1．密码子(codon)2．反义密码子(synonymous codon)3．反密码子(anticodon)4．变偶假说(wobble hypothesis)5．移码突变(frameshift mutant)6．氨基酸同功受体(isoacceptor)7．反义RNA(antisense RNA)8．信号肽(signal peptide)9．简并密码(degenerate code)10．核糖体(ribosome)11．多核糖体(poly some)12．氨酰基部位(aminoacyl site)13．肽酰基部位(peptidy site)14．肽基转移酶(peptidyl transferase)15．氨酰- tRNA 合成酶(amino acy-tRNA synthetase) 16．蛋白质折叠(protein folding)17．核蛋白体循环(polyribosome)18．锌指(zine finger)19．亮氨酸拉链(leucine zipper) 20．顺式作用元件(cis-acting element) 21．反式作用因子(trans-acting factor) 22．螺旋-环-螺旋(helix-loop-helix)第一章 蛋白质1．两性离子：指在同一氨基酸分子上含有等量的正负两种电荷，又称兼性离子或偶极离子。
Rice Genome review :Rice Genome review Sushama Salgaonkar M.Sc.part II 17-09-12Introduction:Introduction The world’s first genome of a crop plant that was completely sequenced Rice-monocarpic annual plant that usually grows between 1 and 1.8 meters tall with long slender leaves 50–100 cm long and 2–2.5 cm broad. Its small, wind-pollinated flowers are characteristic of grasses. The seed is a grain normally 5–12 mm long and 2–3 mm thick The rice genome is well mapped and well characterized, estimated 400 to 430 Mb.Japonica and Indica:Japonica and Indica three main varieties- Oryza sativa : Indica : variety is long-grained, for ex. Basmati rice, grown notably on the Indian sub-continent. Japonica : Japonica rice is short-grained & high in amylopectin (thus becoming "sticky" when cooked), and is grown mainly in more temperate or colder regions such as Japan. Javonica : Javonica rice is broad-grained & grown in tropical climates.Rice is a model cereal plant:Rice is a model cereal plant The small size of its genome (430 Mb) its relatively short generation time its relative genetic simplicity (it is diploid, or has two copies of each chromosome). easy to transform genetically. belongs to the grass family the greatest biodiversity of cereal cropsInstitute which sequence the particular chromosome:Institute which sequence the particular chromosome Sr no. Rice sequence participant Chromosomes 1 Rice Genome R esearch Program (RGP)Japan 1,6,7,8 2 Korea Rice Genome Research Program (Korea) 1 3 CCW(US) CUG(Clemson university) Cold spring Harbor University 3,10 4 TIGR –US 3,10 5 PGIR-US 10 6 University of Wisconsin-US 11 7 National Center of Gene Research Chinese Academy of science -china 4 8 Indian rice genome program-university of Delhi 11 9 Academia sinica plant genomic center (Taiwan) 5 10 Genoscope -France 12PowerPoint Presentation:Sr no. Rice sequence participant Chromosomes 13 Universidad fedral de Pelotas -Brazil 12 14 Kasetsant University –Thailand 9 15 MG Gill University –Canada 9 16 John innescenter –U.K 2Milestone in rice genome sequencing :Milestone in rice genome sequencing 2)Feb 1998 -IRGSP launched under coordination of RGP 1)Sept 1997 – Sequencing of the rice genome was initiated as an international collaboration among 10 countries 5)Dec 2002 – IRGSP finished high-quality draft sequence (clone-by-clone approach) with a sequence length, excluding overlaps, of 366 Mb corresponding to ~92% -RG 3)April 2000 – Monsanto Co. produced a draft sequence of BAC covering 260 Mb of the rice genome; 95% of rice genes were identify identified4)Feb 2001 – Syngenta produced a draft sequence & identified 32,000 to 50,000 genes, 99.8% sequence accuracy & identified 99% of the rice genes 6)Dec 2004-IRGSP produce the high-quality ’ sequence -entire rice genome; with 99.99% accuracy & without any sequence gapIndian complete work on RG sequence :Indian complete work on RG sequence The IRGSP initiated Japan in 1997 and it got under way in 1998. The project estimated to cost about $200 million. India joined -IRGSP in June 2000 and chose to sequence a part of chromosome 11. India has investedRs.48.83 crores for the "Indian Initiative for Rice Genome Sequencing (IIRGS)". The initiative is a joint effort by the Department of Plant Molecular Biology (DPMB), University of Delhi South Campus (UDSC) and the National Research Centre on Plant Biotechnology (NRCPB) and the Indian Agricultural Research Institute (IARI), New Delhi.PowerPoint Presentation:The Indian share of chromosome 11 has been equally divided between these two centres. It was known to carry several diseases resistant gene including in Xac1bacterial blight resistant gene-selected Finding: chromosome segment sequence by IARI involve-6.825 million bp & predicted 1005 gene –with unknown functionMethods:Methods Clone-by-clone Generate a genomic library endonuclease digest Ligation into BACs Transformation in E.coli Collection of clone DNA fragment result in genomic DNA libraryPowerPoint Presentation:Shotgun sequencing Genome is broken into much smaller, overlapping fragment . Each fragment is sequenced Genome is assembled on overlapping sequencing Useful for sequencing small genomeStructure of rice chromosome:Structure of rice chromosomeObservation Table :Observation Table Organism type relevance Genome size Chromosome no. No. of predicted gene Organisation Year of completion Sequence method done Rice Oryza sativa. J aponica nipponbare Crop & model plant 466MB 12 35,000-50,000 IRGSP 2002 Shot gun Rice Oryza sativa . indica Crop 420MB 12 46,022-55,615 Beijing genomic institute 2002 Whole genome shot gunSignificant findings from the sequencing of rice genome :Significant findings from the sequencing of rice genome The rice genome is about 389 mb , 370.7 mb has been sequenced, 18.1 mb unsequenced . Sequenced segment represents 99% of euchromatin and 95% of rice genome The rice genome has -40,000genes (37,344 coding genes) One gene can be found every 9.9 kb, a lower density than that observed in Arabidopsis. 29% in clustered gene families 71% putative homology with Arabidosis , 90% Arabidopsis genes have putative homologue with rice. 2,859 genes unique to rice and other cerealsApplication :Application Understanding-plant evolution & the differences between monocots & dicots Improve-efficiency of rice breeding. Improve-nutritional value of rice, enhance crop yield by improving seed quality, resistance to pests and diseases & plant hardiness Development of gene-specific markers for marker-assisted breeding of new and improved rice varieties How a plant responds to the environment and which genes control various functions-plantPowerPoint Presentation:Repetitive DNA is estimated to constitute at least 50% of the rice genomeReferences:References /pmc/articles/PMC526008 .www ./ downlaod /publication /list/en/61.pdf /genome/10/fl1911/19110810.htm/wiki/Transposable_element。
Multiple Choice(1) The attachment site for RNA polymerase in bacteria is called the:a. Initiatorb. Operatorc. Promoterd. Start codon(2) The specificity of bacterial RNA polymerase for their promoters is due to which subunit?a. αb. βc. γd. σ(3) The first protein complex to bind to the core promoter for a protein-coding gene in eukaryotes is;a. RNA polymerase IIb. General transcription factor TFIIBc. General transcription factor TFIIDd. General transcription factor TFIIE(4) Which modification must be made to RNA polymerase II in order to activate the preinitiation complex?a. Acetylationb. Methylationc. Phosphorylationd. Ubiquitination(5) What is the name of the DNA sequence that is located near the promoter of the lactose operon, and which regulates expression of the operon in E. coli?a. Activatorb. Inducerc. Operatord. Repressor(6) Which of the following types of sequence module enables transcription to respond to general signals from outside of the cell?a. Cell-specific modulesb. Developmental modulesc. Repression modulesd. Response modules(7) Which of the following is NOT a type of activation domain?a. Acidic domainsb. Glutamine-rich domainsc. Leucine-zipper domainsd. Proline-rich domains(8) Which of the following is NOT a experiment used to define the site on a DNA molecule to which a protein binds?a. Gel retardation assayb. DNA footprinting assayc. Modification interference assayd. Y east two hybrid assay(9) Which of the following DNA sequences can increase the rate of transcription initiation of more than one gene/promoter?a. Activatorsb. Enhancersc. Silencersd. T erminators(10) Approximately how many base pairs form the attachment between the DNA template and RNA transcript during transcription?a. 8b. 12-14c. 30d. The entire RNA molecule remains base-paired to the template until transcription is finished.(11) Which factor is thought to be most important in determining whether a bacterial RNA polymerase continues or terminates transcription?a. Nucleotide concentrationb. Structure of the polymerasec. Methylation of termination sequencesd. Thermodynamic events(12) What is the role of the Rho protein in termination of transcription?a. It is a helicase that actively breaks base pairs between the template and transcript.b. It id s DNA-binding protein that blocks the movement of RNA polymerase along the template.c. It is a subunit of RNA polymerase that binds to RNA hairpins and stalls transcription.d. It is a nuclease that degrades the 3’ ends of RNA transcripts.(13) Antitermination is involved in regulation of which of the following?a. Operons encoding enzymes involved in the biosynthesis of amino acids with regulation dependent on the concentration of the amino acids.b. Operons encoding enzymes involved in the degradation of metabolites, regulation dependent on the presence of the metabolitec. Genes present in the upstream region of the operond. Genes present in the downstream region of the operon.(14) What is the major transcriptional change that occurs during the Stringent Response in E. coli?a. Transcription rates are increased for most genes.b. Transcription rates are increased only for the amino acid biosynthesis operons.c. Transcription rates are decreased for most genes.d. Transcription rates are decreased only for the amino acid biosynthesis operons.(15) Which of the following is necessary for the RNA endonuclease activity of RNA polymerase that occurs when RNA polymerase is stalled during transcription?a. Rhob. RelAc. GreAd. RNAse H(16) How is the lariat structure formed during splicing of a GU-AG intron?a. After cleavage of the 5’ splice site, a new phosphodiester bond is formed between the 5’ nucleotide and the 2’ carbon of the nucleotide at the 3’ splice site.b. After cleavage of the 5’ splice site, a new phosphodiester bond is formed between the 5’ nucleotide and the 2’ carbon of an internal adenosine.c. After cleavage of the 5’ splice site, a new phosphodiester bond is formed between the 5’ nucleotide and the 2’ carbon of the nucleotide at the 5’ splice site.d. After cleavage of the 3’ splice site, a new phosphodiester bond is formed between the 5’ nucleotide and the 2’ carbon of an internal adenosine.(17) What are cryptic splice sites?a. These are splice sites that are used in some cells, but not in others.b. These are splice sites that are always used.c. These are splice sites that are involved in alternative splicing, resulting in the removal of exons from some mRNA molecules.d. These are sequences within exons or introns that resemble consensus splicing signals, but are not true splice sites.(18) What statement correctly describes trans-splicing?a. The order of exons within an mRNA transcript is rearranged to yield a different mRNA sequence.b. Exons are deleted from some mRNA transcripts but not others.c. Intron sequences are not removed from RNA transcripts and are translated into proteins.d. Exons from different RNA transcripts are joined together.(19) The chemical modification of eukaryotic rRNA molecules takes place in the:a. Cytoplasm.b. Endoplasmic reticulum.c. Nuclear envelope.d. Nucleolus.(20) Which of the following is an example of RNA editing?a. Removal of introns from an RNA transcript.b. Degradation of an RNA molecule by nucleases.c. Alteration of the nucleotide sequence of an RNA molecule.d. Capping of the 5’ end of an RNA transcript.(21) Nonsense-mediated RNA decay (NMD) is a system for the degradation of eukaryotic mRNA molecules with what features?a. NMD degrades mRNA molecules with stop codons at incorrect positions.b. NMD degrades mRNA molecules that encode nonfunctional proteins.c. NMD degrades mRNA molecules that lack a start codon.d. NMD degrades mRNA molecules that lack a stop codon.(22) Which of the following describes RNA interference?a. Antisense RNA molecules block translation of mRNA molecules.b. Double-stranded RNA molecules are bound by proteins that block their translation.c. Double-stranded RNA molecules are cleaved by a nuclease into short interfering RNA molecules.d. Short interfering RNA molecules bind to the ribosome to prevent the translation of viral mRNAs.(23) How are RNA molecules transported out of the nucleus?a. Passive diffusion through the membrane.b. Through the membrane pores in an energy-dependent process.c. Through membrane pores in an energy independent process.d. Through a channel in the membrane that leads to the endoplasmic reticulum.(24) Match protein/RNA with its function (answers can be used more than once or not at all)Spliceosome a. small nuclear ribonucleoproteins (snRNP)microRNAs b. guanylyl transferasemRNA capping c. ribozymeautocatalytic RNA splicing d. dicere. poly-A polymerase(25) Match protein with its function (answers can be used more than once or not at all)JAK a. G-proteinGlucocorticoid receptor b. EndonucleaseRAS c. DNA-binding proteinIF-2 d. RNA binding proteine. Kinase(26) Match lambda gene with its function (answers can be used more than once or not at all)cI a. Anti-terminationN b. Transcriptional repressorCRO c. Transcriptional activatorcII d. Transcriptional terminatore. Translation factorAnswers to practice exam #3How is it possible for microRNAs to regulate eukaryotic gene expression by binding to the 3’ untranslated end of an mRNA ?Binding to the 3’-UTR initiates an RNA cleavage event that removes the polyA tail and begins the mRNA degradation processWhy is attenuation absent in eukaryotic organisms ?Attenuation is the mechanism whereby amino acid biosynthesis operons are regulated by the cellular concentration of the amino acid that is the product of the genes in the operon by transcription termination. The attenuation mechanism requires that translation by ribosomes and transcription occur in the same subcellular compartment. In eukaryotes transcription and translation are carried out in different compartments, so attenuation would not be possible in eukaryo tes.What are the differences between activator and coactivator proteins ?An activator is a DNA binding protein that stabilizes construction of the RNA polymerase II transcription initiation complex. A coactivator is a protein that stimulates transcription initiation by binding nonspecifically to DNA or via protein-protein interactions.Explain what a “modification protection assay” is intended to discover and how it is carried out .Modification protection is a technique used to identify nucleotides i nvolved in interactions with a DNA-binding proteinHow are Caenorhabditis elegans and Drosophila melanogaster good model organisms for development in higher eukaryotes ?Developmental pathways in animals utilize similar regulators, therefore discovery of regulators in lower animals can reveal how development is controlled in higher animals.How does the anchor cell of C. elegans induce the vulva progenitor cells to differentiate into vulva cells? Why do the vulva progenitor cells follow different pathways upon receiving the signal from the anchor cell ?The anchor cell produces a diffusible signal that stimulates differentiation of vulva cells. Different vulva cells undergo different differentiation pathways because they are exposed to differing concentr ations of the signal molecule, and the vulva cells themselves produce secondary signaling molecules that control differentiation in nearby vulva cells.The process of excision of a GU-AG intron and splicing of exons is defined as requiring two transesterification reactions. What does this mean ?A transesterification reaction is the simultaneous cleavage and reformation of a phosphodiester bond. During intron splicing the donor site phosphodiester bond is cleaved and then reformed with the branchpoint nucleotide within the intron, forming a lariat structure. In the second transesterification, the branch point phospodiester bond I cleaved and simultaneously formed between the donor and acceptor sites. The net effect is that there is no change in the number of phosphodiester bonds. During sporulation in Bacillus σE and σF are present in both the prespore and mother cells. How is σF activated in the prespore?Sigma F is activated in the prespore by when it is released from protein-protein interaction with AB. Sigma F is inactive when it is bound to AB.Explain how the iron response protein (IRP) functions to activate expression of Ferritin and at the same time inhibit expression of Transferrin.The iron response protein can bind to iron response elements in RNA only when it is not bound to iron. In the case of ferritin, binding of IRP to the 5’-IRE blocks translation of the ferritin mRNA, so when it is not bound ferritin protein is produced. In the case of transferrin, binding to the 3’-IRE blocks degradation of the transferrin mRNA thereby increasing half life of the mRNA and stimulating transferrin protein production.。
33. Directions: Reorder the disordered parts of a sentence to make a complete sentence.1) Facebook allows anyone who declares themselves to be aged 13 or older to become amember of the website.2) The website's membership was initially limited by the founders to Harvard students, but wasexpanded to other colleges in the Boston area, the Ivy League, and Stanford University.3) Facebook later expanded membership eligibility to employees of several companies,including Apple Inc. and Microsoft.4) The media often compares Facebook to MySpace, but one significant difference between thetwo websites is the level of customization.5) The patent may encourage Facebook to pursue action against websites that violate the patent,which may potentially include websites such as Twitter.4. Directions: Change the following sentences into nominalized ones.1) Network security has become a major concern for organizations and individuals.2) For these reasons, making an operating system ready and available is helpful.3) The security policy development process may also involve the identification of protection.4) Rather, it is a brief description of a number of important product features, such as the inputmethod, network connectivity, operating system, and database capabilities.5) While formulating the interview plan, designers should work closely with projectstakeholders who have access to users.Unit 43. Directions:1)Given that robots generally lack muscles, they can't rely on muscle memory (the trickthat allows our bodies to become familiar over time with movements such as walking orbreathing) to help them more easily complete repetitive tasks.2)For autonomous robots, this can be a bit of a problem, since they may have toaccommodate changing terrain in real time or risk getting stuck or losing their balance.3)One way around this is to create a robot that can process information from a variety ofsensors positioned near its “legs” and identify different patterns as it moves, a team ofresearchers report Sunday in Nature Physics.4)Some scientists rely on small neural circuits called "central pattern generators" (CPG) tocreate walking robots that are aware of their surroundings.5)Bernstein Center for Computational Neuroscience researcher Poramate Manoonpongand Max Planck Institute for Dynamics and Self-Organization researcher Marc Timmeare leading a project that has created a six-legged robot with one CPG that can switchgaits depending upon the obstacles it encounters.4. Directions: Change the following sentences into the passive-voice ones.1) A novel twist is introduced on this traditional approach (by the new field of experimentalphilosophy).2)T he search to understand people’s ordinary intuitions is continued (by experimentalphilosophers).3)This is accomplished by using the methods of contemporary cognitive science—experimental studies, statistical analyses, cognitive models, and so forth.4)Just in the past year or so, this new approach has been being applied (by a number ofresearchers) to the study of intuitions about consciousness.5)How people think about the mind can be better understood by By studying how peoplethink about three different types of abstract entities.Unit 7 Genetic Engineering2. Directions: Complete the sentences with the words given in the brackets. Change the form ifnecessary.1) Restriction enzyme is an endonuclease that cuts DNA at sites defined by its recognitionsequence.DNA限制内切酶是一种酸内切酶，它能够在其识别序列指定的部位切割DNA。
Molecular CellShort ArticleStructure of the Endonuclease Domainof MutL:Unlicensed to CutMonica C.Pillon,1Jessica J.Lorenowicz,1Michael Uckelmann,2Andrew D.Klocko,3Ryan R.Mitchell,1Yu Seon Chung,1 Paul Modrich,4,5Graham C.Walker,6Lyle A.Simmons,3Peter Friedhoff,2and Alba Guarne´1,*1Department of Biochemistry and Biomedical Sciences,McMaster University,Hamilton,ON L8N3Z5,Canada2Institut fu¨r Biochemie(FB08),Justus-Liebig-Universita¨t,D-35392Giessen,Germany3Department of Molecular,Cellular,and Developmental Biology,University of Michigan,Ann Arbor,MI48103,USA4Department of Biochemistry5Howard Hughes Medical InstituteDuke University Medical Center,Durham,NC27710,USA6Department of Biology,Massachusetts Institute of Technology,Cambridge,MA02139,USA*Correspondence:guarnea@mcmaster.caDOI10.1016/j.molcel.2010.06.027SUMMARYDNA mismatch repair corrects errors that have escaped polymerase proofreading,increasing repli-cationfidelity100-to1000-fold in organisms ranging from bacteria to humans.The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonu-clease domain of Bacillus subtilis MutL.The structure is organized in dimerization and regulatory subdo-mains connected by a helical lever spanning the conserved endonuclease motif.Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mecha-nism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL.The structure also provides a molecular frame-work to propose and test additional roles of MutL in mismatch repair.INTRODUCTIONDNA mismatch repair(MMR)maintains genomic stability by correcting errors that have escaped polymerase proofreading (Kunkel and Erie,2005).MMR proteins are also implicated in a variety of other cellular processes such as DNA damage signaling,apoptosis,meiotic and mitotic recombination,and somatic hypermutation(Modrich,2006).Mutations in MMR genes are associated with an increased mutation rate and micro-satellite instability,the hallmark of human nonpolyposis colo-rectal cancer(Peltomaki,2005).Initiation of MMR depends on the coordinated action of three proteins.MutS recognizes a mismatched base pair or a small insertion/deletion loop and recruits MutL in an ATP-dependent manner.Subsequently,the newly synthesized strand is marked for repair.In Escherichia coli,strand discrimination is achieved by mismatch-provoked activation of the MutH endonuclease, which cleaves the unmethylated DNA strand at hemimethylated GATC sites transiently generated during DNA replication. Although most bacteria and all eukaryotes do not encode a MutH homolog,a pre-existing nick is sufficient to activate MMR in a system reconstituted from purified proteins(Zhang et al.,2005).It has been shown that MutL homologs from species lacking a MutH endonuclease harbor an intrinsic latent nicking endonuclease activity that is vital for its function in MMR(Erdeniz et al.,2007;Kadyrov et al.,2006,2007,2008).MutL is composed of two structurally conserved domains connected by a variableflexible linker(Guarne´et al.,2004). The N-terminal region encompasses an ATPase domain of the GHL ATPase superfamily that is conserved from bacteria to humans(Ban et al.,1999;Guarne´et al.,2001).Conversely,the sequence conservation in the C-terminal dimerization region of MutL is low.The structure of the C-terminal domain of E.coli MutL(EcMutL)reveals that this region is organized into two distinct subdomains(Guarne´et al.,2004;Kosinski et al., 2005).While prokaryotic MutL homologs form homodimers,their eukaryotic counterparts form heterodimers.In humans,there are four paralogs of MutL(hMLH1,hPMS2,hPMS1,and hMLH3)that form three heterodimers by association of hMLH1 with hPMS2(hMutL a),hPMS1(hMutL b),and hMLH3(hMutL g) (Li and Modrich,1995;Lipkin et al.,2000;Raschle et al.,1999). hMutL a is necessary for MMR function,and hMutL g has a role in meiotic recombination;however,the function of hMutL b is unknown(Kunkel and Erie,2005).The C-terminal regions of hPMS2and hMLH3encompass a conserved DQHA(X)2E(X)4E motif that is required for endonuclease activity.Based on sequence analysis and molecular modeling,three additional conserved motifs(ACR,C[P/N]HGRP,and FXR)have been pre-dicted to form a single active site with the endonuclease motif (Kosinski et al.,2008).Analysis of the reconstituted human MMR system indicates that the endonuclease activity of MutL a provides a loading site for MutS a-activated exonuclease I (Kadyrov et al.,2006).Molecular Cell39,145–151,July9,2010ª2010Elsevier Inc.145Here we present the structure of the C-terminal dimerizationdomain of Bacillus subtilis MutL (BsMutL)harboring the endo-nuclease activity of the protein.The structure reveals the conserved three-dimensional organization of the endonuclease site of MutL and exposes the presence of a regulatory Zn 2+-binding site that is important for the MMR function of BsMutL in vivo.The structure allows for us to propose a model describing how the association of MutS and the DNA polymerase III proces-sivity clamp (b clamp)with MutL could license nicking of a newly synthesized DNA strand.RESULTS AND DISCUSSIONCrystal Structure of BsMutL-CTDThree crystal forms of the C-terminal domain of BsMutL (BsMutL-CTD)were obtained.Crystal form I was used to deter-mine the structure of BsMutL-CTD by multiwavelength anoma-lous diffraction using crystals grown with Sel-Met substituted protein (Table 1).This crystal includes four independent mono-mers (molecules A–D)in the asymmetric unit that associate through crystal symmetry to form the functional BsMutL-CTD dimer.Crystal forms II and III contained a single dimer in the asymmetric unit.In the three structures,the N-and C-terminal ends of BsMutL-CTD (residues 433–461/580–627)define the dimerization subdomain,while residues 474–573define an external subdomain that protrudes to the solvent,herein referred to as the regulatory subdomain (Figures 1A and 1B).The subdo-mains are connected by helix a A (residues 463–473),encom-passing the conserved endonuclease motif,and the linker con-necting helices a D-a E (residues 575–581),which is disordered in our structures (Figure 1A).However,the relative orientation between subdomains varies from one crystal form to another (see Movie S1available online).The three complimentary conserved motifs associate with the endonuclease motif cluster around helix a A to delineate a single catalytic site with the conserved endonuclease motif (462DQHA(X)2E(X)4E)(Figures 1B and 1C).604CPHGRP resides in the a E-b 8loop,572SCK (consensus sequence ACR)is the last turn of the a D helix,and 623FKR,at the C terminus of the protein,reaches the active site of the other protomer (Figure 1B).Except for the 572SCK motif,contributed by the regulatory subdomain,all motifs reside in the dimerization subdomain.Two additional conserved motifs have been identified within the C-terminal region of MutL a (Kosinski et al.,2008).The 443GQ motif resides on the b 1strand of the dimerization subdo-main.This strand is in the vicinity of the a D-a E loop and hence it may indirectly contribute to the overall stability of the active site.The 487QEMIVP motif (consensus sequence QXLLXP)is on the surface of the regulatory subdomain and conspicuously exposed to the solvent (Figure 1B).Conservation of the QXLLXP motif is not correlated with the endonuclease activity of MutL,suggesting that it could mediate the interaction with other repair factors.Even though the C-terminal regions of EcMutL and BsMutL have very low sequence similarity,their structures have nearly identical topologies (Figure S1).However,key differences exist.Superimposition of the EcMutL-CTD monomer onto the BsMutL-CTD monomer returned root-mean-square deviations (rmsds)of only 1A˚for the dimerization subdomains but >2A ˚forthe Table 1.Data Collection and Refinement Data Collection Crystal Form I Crystal Form II Crystal Form III Space Group C2221P212121P212121Molecular CellStructure of the Endonuclease Domain of MutL146Molecular Cell 39,145–151,July 9,2010ª2010Elsevier Inc.regulatory subdomains,reflecting the increased divergence of this region.This is intriguing because the regulatory subdomain contributes minimally to the endonuclease site.The most striking difference between the two structures is the organization of the secondary structure elements surrounding helix a A,which would preclude the formation of a functional endonuclease site even if EcMutL had the conserved DQHA(X)2E(X)4E motif (Figure S1).Notably,the extended a E-b 8loop in BsMutL,rather than the additional helix seen in EcMutL,brings the 604CPHGRP motif closer to helix a A and secludes the endonuclease site.The dimerization interfaces are also remarkably different.While theBsMutL dimer buries 1065A˚2,the EcMutL dimer only conceals 910A˚2.Interestingly,the reorientation of the apposing b sheets in the BsMutL-CTD dimer allows the 623FKR motif to reach the adjacent endonuclease site.BsMutL Has Weak Endonuclease ActivitySimilarly to other MutL orthologs,BsMutL had a weak endonu-clease activity dependent on Mn 2+(Figure 2A).Both a point mutation in the endonuclease motif (D462N)and deletion of the ATPase domain virtually abolished the endonuclease activity of BsMutL (Figure 2A).This finding is interesting in light of the recent experiments revealing that the C-terminal domains of Neisseria ghonorrhoeae and Aquifex aeolicus have endonuclease activity (Duppatla et al.,2009;Mauris and Evans,2009).However,the specific activity of AaeMutL-CTD is much lower than that of the full-length protein.We suspected that the lack of nicking activity by BsMutL-CTD could be due to a DNA-binding defect,since EcMutL-CTD does not bind DNA stably (Guarne ´et al.,2004).Indeed,BsMutL-CTD did not bind supercoiled DNA,whileother variants of BsMutL did (Figure 2C).Addition of 0.5mM ATP stimulated the nicking activity of BsMutL,but higher concentrations of ATP (5mM)inhibited the nicking activity,presumably due to excess nucleotide chelating Mn 2+ions away (Figure 2B,lanes 5and 6).Unexpectedly,addi-tion of ATP and/or Mg 2+stimulated a second cut on the nicked DNA to yield a linear product.The cut of the two strands at nearby points could be due to the presence of two endonuclease sites in the BsMutL homodimer or a consequence of the high-ion concentrations used in the experiment.We favor the former because incubation with 10mM Mn 2+did not cause nicking of the two strands (data not shown),but addition of only 1mM of a second metal ion such as Zn 2+or Co 2+yielded a linear product (Figure 2D).Interestingly,Mg 2+did not support double nicking under these conditions,suggesting that BsMutL may have higher affinity for Zn 2+or Co 2+than Mg 2+.We then characterized the ATPase activity of BsMutL (K m =0.4mM and k cat =0.3min À1)and found that it is a weakerATPase than other MutL homologs (Ban et al.,1999;Guarne´et al.,2001;Hall et al.,2002).Given the slow ATP-hydrolysis rate,the stimulation of the endonuclease activity of BsMutL was likely due to ATP binding rather than ATP hydrolysis.In good agreement with this idea,ADP did not stimulate the endonuclease activity of BsMutL (Figure 2B,lanes 9and 10).However,two known nonhydrolyzable analogs of ATP,AMPPnP and ATP g S,did not stimulate the endonuclease activity of BsMutL beyond the levels observed when both Mn 2+and Mg 2+were present (Figure 2B,compare lanes 4,7,9,and11).Figure 1.Crystal Structure of BsMutL-CTD(A)Ribbon diagram of the BsMutL-CTD monomer.Secondary structure motifs are labeled and colored blue (helices)and yellow (strands)with the connecting loops in light green.The endonuclease and the endonuclease-associated motifs are shown in purple,while the additional conserved motifs are shown in orange.(B)Ribbon diagram of the BsMutL-CTD dimer with one protomer shown as in (A)and the other one as gray ribbons.(C)Sequence alignment of the C-terminal regions of BsMutL,hPMS2,and EcMutL.Secondary structure elements of BsMutL-CTD are shown as arrows (strands)and cylinders (helices).The five conserved motifs are highlighted in purple and underlined.Conserved hydrophobic residues are highlighted in yellow.The conserved 487QEMIVP motif is highlighted in orange.See also Figure S1.Molecular CellStructure of the Endonuclease Domain of MutLMolecular Cell 39,145–151,July 9,2010ª2010Elsevier Inc.147Conceivably,the ATP-dependent stimulation of the endonu-clease activity of BsMutL could be due to a conformational change induced by ATP binding as seen in other MutL orthologs (Ban et al.,1999;Sacho et al.,2008),which would bring DNA bound at the ATPase domain in close proximity to the endonu-clease site.If this is the case,our results suggest that only ATP can induce efficiently such conformational change.BsMutL Has a Regulatory Zn 2+-Binding SiteAlthough the endonuclease activity of BsMutL was metal depen-dent and the conserved motifs around helix a A define a putative Zn 2+-binding site (Kosinski et al.,2008),no metal ions were found in the BsMutL-CTD structure (crystal form I).However,different crystal forms were obtained when the protein storage buffer (crystal form II)and the crystallization solution (crystal form III)were supplemented with ZnCl 2.While the 462DQHA(X)2E(X)4E,572SCK,and 604C(P/N)HGRP motifs were much closer in crystal form II than crystal form I,no metal was found in this crystal form either.Conversely,the BsMutL-CTD dimer found in the asym-metric unit of crystal form III contained two Zn 2+ions bound to each protomer;a fully occupied Zn 2+ion (Zn 2+A)was coordi-nated by the side chains of residues Glu468,Cys604,His606,and a well-ordered water molecule,and a partly occupied site (Zn 2+B)coordinated by the side chains of residues His464,Glu468,Cys573and a water molecule (Figure 3A).The nature of the metal ion was confirmed on the anomalous difference electron density maps from diffraction data collected at the Zn 2+absorption edge (Table 1).It had been previously reported that the putative Zn 2+-binding site in hPMS2could be related to the regulatory metal-binding site found in the iron-dependent repressors from the DtxR/MntR family (Kosinski et al.,2008).A structural comparison revealed that,while motifs 462DQHAX 2EX 4E,572SCK,and 604CPHGRP from BsMutL could be superimposed to the regula-tory metal-binding sites of IdeR or MntR,the residues coordi-nating the metal ion differed.However,another Mn 2+-dependent repressor from the same family (ScaR,PDB 3HRU)encom-passes a regulatory metal-binding site identical to the fully occu-pied Zn 2+site in BsMutL-CTD.To probe whether the two metal-bound sites found in our structure were true Zn 2+-binding sites,we measured the affinity of purified BsMutL-CTD and various BsMutL-CTD variants for zinc.To this end,we measured the fluorescence of increasing concentrations of Zn 2+bound to the fluorescence indicator FluoZin-3.In the absence of protein,fluorescence increased exponentially,reaching maximum values at around 1.5–2m M ZnCl 2.The sigmoidal response observed for wild-type and the D462N,H464S,and E473K variants was characteristic of zinc binding by the protein (Figure 3B),suggesting that these BsMutL-CTD variants still retained the ability to bind zinc.Addi-tion of the sulfhydryl-modifying agent methyl methanethiosulfo-nate (MMTS)resulted in an increase of fluorescence to the level detected in the absence of protein,indicating that one or more cysteine residues within the C-terminal domain of BsMutL were important for Zn 2+binding (data not shown).Conversely,fluorescence profiles of E468K,C604A,H606S,and C604A/H606S mutants did not have a sigmoidal response (Figure 3B),revealing that these BsMutL-CTD variants had lost the ability to bind zinc.These results confirmed that Glu468,Cys604,and His606define the Zn 2+-binding site in BsMutL (Figure 3A).Integrity of the Conserved Motifs Is Important for Mismatch Repair In VivoWe presumed that the integrity of the Zn 2+-binding site in MutL would be important for proper MMR.Therefore,we measured the MMR efficiency of BsMutL variants encompassing point mutations in the conserved residues involved in the endonu-clease or Zn 2+-binding sites.Mutation of D462A,H464A,E468K,or H606A completely inactivated MMR in vivo (Figure 3C),under-scoring the importance of these residues.Similarly,the equiva-lent mutations in hPMS2also conferred a strong mutator pheno-type (Kosinski et al.,2008).Conversely,the BsMutL-Q463A and BsMutL-E473K variants had similar MMR efficiency to wild-type BsMutL,suggesting that not all the conserved residues within these motifs play essential roles in MMR.We next analyzed the importance of other conserved motifs found in the C-terminal region of MutL,namely the 487QEMIVPFigure 2.Endonuclease Activity of BsMutL(A)Nicking activity of BsMutL (left)and BsMutL-CTD (center)in the presence of Mg 2+,Zn 2+,Mn 2+,or Cd 2+as parison of the nicking activity of BsMutL and BsMutL-D462N in the presence of Mn 2+(right).Migration of supercoiled (SC),nicked (N),and linear (L)DNA is indicated.(B)Endonuclease activity of BsMutL in the presence of 0.5mM (+)and 5mM (++)nucleotide.(C)DNA binding by BsMutL (WT),BsMutL-CTD (CTD),and BsMutL variants as indicated.Data are presented as the mean of three independent measure-ments,and the error bars correspond to the standard errors of the mean (SEM =s /O n,where s is the average and n the sample size).(D)Stimulation of the endonuclease activity of BsMutL (1mM Mn 2+)by a second divalent metal ion (1mM).Molecular CellStructure of the Endonuclease Domain of MutL148Molecular Cell 39,145–151,July 9,2010ª2010Elsevier Inc.motif.Mutation of Ile490almost completely inactivated MMR in vivo,whereas mutation of Pro492was without effect (Fig-ure 3C).A BsMutL-Q487A variant conferred approximately a 50-fold mutator phenotype about 5-fold lower than a mutL null strain.Replacement of residues 487QEMIV with five alanine residues also abrogated MMR in vivo (Figure 3C).This conserved loop is conspicuously exposed and loosely resembles the consensus b -binding motif (Dalrymple et al.,2001).Most notably,its conformation is nearly identical to that seen in the structures of other peptides bound to the b clamp (Figure 3D).Superimposition of the 486VQEMIVPL sequence from BsMutL onto the structures of b clamp bound to peptides from polymerase II,FEN-1,andpolymerase IV returned rmsds smaller than 0.5A˚.Accordingly,the regulatory subdomains of both BsMutL and EcMutL could be directly docked onto the structure of the E.coli b clamp.Inter-estingly,docking of the MutL-CTD dimer suggests that the inter-action of one protomer would prevent the interaction of the other due to steric hindrance (Figure S2),suggesting a possible regula-tory role for this interaction.A Model for the Activation of the Endonuclease Activity Based on the structures of BsMutL-CTD,we suggest that the conserved motifs in MutL define an endonuclease active site with two distinct subsites:a structural Zn 2+-binding site defined by the side chains of Glu468,Cys604,and His606;and a catalytic site likely defined by Asp462and His464.Metal binding at the structural site locks the orientation between the dimerization and regulatory subdomains,which is highly variable in the absence of metal (Movie S1).Based on the three-dimensional organization of Asp462and His464,the catalytic subsite could coordinate one or two metal ions to nick DNA (Yang,2008).Sup-porting this idea,double-stranded DNA could be modeled onto the structure of BsMutL-CTD with the scissile bond at a distance compatible with catalysis activated by Asp462and the adjacent 30phosphate providing the fourth coordination ligand of the regulatory Zn 2+ion (Figure S3).In the context of the replication fork,the endonuclease activity of MutL ought to be repressed until a mismatch is encountered.A look at the electrostatic potential surface of BsMutL-CTD reveals a powerful suppression mechanism of DNA binding.The regulatory subdomain is covered with negative charges that guard the endonuclease site (Figure 4A).This could be a widely spread repression mechanism,since some of the nega-tively charged residues in helix a C are conserved in other MutL homologs harboring the endonuclease motif (Figure 1C and Kosinski et al.,2008).Licensing the endonuclease activity of MutL would thus require a significant conformational change or the interaction with other repair factors to overcome the DNA repulsion in the vicinity of the endonuclease site.We presume that a conformational change like that induced in MutL a upon nucleotide binding could allow DNA bound at the ATPase domain to reach the endonuclease site (Sacho et al.,2008).However,additional repair factors are likely required to mask helix a C.The endonuclease activity of MutL a is greatly stimulated by the presence of PCNA and RFC (Kadyrov et al.,2006;Kadyrov et al.,2007),the eukaryotic homologs of the b clamp and the clamp loader.Additionally,MutS a and PCNA form a stable complex (Iyer et al.,2008).Human MutL a interacts with MutS a through its ATPase domain (Plotz et al.,2006),but the region of MutS a that interacts with MutL a is not known.Conceivably,the three proteins could form a ternary complex involved in strand discrimination;however,whether MutS a and MutL a can interact simultaneously with PCNA is controversial (Dzantiev et al.,2004;Lee and Alani,2006).Bacterial MutS has twobindingFigure 3.Regulatory Zn 2+-Binding Site in BsMutL-CTD(A)Organization of the endonuclease site of BsMutL-CTD bound to Zn 2+(crystal form III).Hydrogen bonds are shown as black dashed lines with the water molecules and Zn 2+ions shown as red and lilac spheres,respectively.Conserved motifs are color coded green (462DQHAX 2EX 4E),purple (604CPHGRP),and orange (572SCK).(B)Zinc-affinity profiles of BsMutL-CTD and point mutants of BsMutL-CTD as indicated.Data are presented as the mean of three independent measurements ±SEM.(C)Bar diagram showing the relative mutation frequency of the indicated mutL variants altered in the Zn 2+-binding,the endonuclease site,and the putative b -binding motif.Data are presented as the mean of four independent cultures ±SEM.(D)Superimposition of the b -binding motif in BsMutL-CTD (green)onto those of Pol II (yellow,PDB 3D1E),Pol IV (cyan,PDB 1UNN),and FEN-1(purple,PDB 1RXM)shown as a main-chain trace with the b clamp structure (PDB 3D1E)presented as a semitransparent electrostatic potential sur-face.See also Figures S2and S3and Movie S1.Molecular CellStructure of the Endonuclease Domain of MutLMolecular Cell 39,145–151,July 9,2010ª2010Elsevier Inc.149sites for the b clamp (Lopez de Saro et al.,2006;Simmons et al.,2008).In B.subtilis ,the C-terminal site is necessary to recruit MutL to mismatches and to activate the MMR response (Simmons et al.,2008).We presume that the ATPase domain of MutL could interact with MutS,while its C-terminal domain interacts with the b clamp (Figure 4B).This model is supported by the presence of the b -binding-like motif (487QEMIV)within the C-terminal domain of MutL and the fact that a PCNA-binding sequence has been identified in the dimerization region of yeast MLH1(Lee and Alani,2006).Collectively,our data pose an attractive model in which the endonuclease activity of MutL is repressed by impaired DNA binding.Based on these data,the simplest mechanism would harness MutL and b clamp,allowing for DNA binding and licensing of the endonuclease activity.Consequently,the struc-ture provides a platform for future mechanistic studies of MutL-MutS-b at the early steps of MMR.EXPERIMENTAL PROCEDURESCloning,Purification,and CrystallizationFull-length BsMutL was amplified from genomic DNA and cloned into the pProEXHTa expression vector (Life Technologies).His-tagged BsMutL was purified using a Ni 2+-chelating affinity column equilibrated with 20mM Tris (pH 8),0.5M NaCl,1.4mM b -mercaptoethanol,5%glycerol,and 100mM PMSF.BsMutL was eluted using 300mM imidazole and subsequently injected on a hydrophobic column equilibrated with 20mM Tris (pH 8),1M KCl,1mM DTT,and 5%glycerol.BsMutL was further purified by ionic exchange and size exclusion chromatography (MonoQ 5/50and Superdex-S200,GE Healthcare)equilibrated with 20mM Tris (pH 8),100mM KCl,1mM DTT,and 5%glycerol (storage buffer).Guided by a structure-based sequence alignment,we sub-cloned the C-terminal fragment of BsMutL (BsMutL-CTD,residues 433–627).BsMutL-CTD was purified similarly to BsMutL with an additional ionic exchange purification step after His-tag cleavage with TEV-protease.Mutants of BsMutL and BsMutL-CTD were generated by QuikChange (Stratagene)and verified by DNA sequencing (MOBIX,McMasterUniversity).Figure 4.Model of Activation of the Endo-nuclease Activity of MutL(A)Orthogonal views of the electrostatic surface potential of the BsMutL-CTD protomer.The second protomer is shown as a ribbon diagram with the endonuclease motifs in purple.(B)Upon mismatch binding,MutS (blue)undergoes a nucleotide-dependent conforma-tional change that triggers recruitment of MutL (green)to the mismatch site,likely aided by the b clamp (purple).ATP binding by MutL then promotes the association of its two ATPase subunits and brings the ATPase in close proximity to the dimerization domain of the protein.Coordi-nated interaction of MutS and b clamp bound to DNA (ribbon diagram)with ATP-bound MutL could thus license the latent endonuclease activity of MutL.ATP and ADP are shown as yellow and orange stars,respectively.Crystal form I was grown in 25%PEG-mono-methyl ether 550,0.1M MgCl 2,0.1M Tris (pH 9),and 5%PEG 400.Two additional crystal forms were obtained when the protein was supple-mented with 50nM ZnCl 2and 50nM CoCl 2.Crystal form II was grown in 25%PEG 3350,0.15–0.2M NaCl,and 0.1M Tris (pH 7).Addition of 0.5mM ZnCl 2to this crystallization solution yielded crystal form III.PEG 400(10%)was added to all crystallization conditions prior to flash freezing in liquid nitrogen.Data Collection and Structure DeterminationFor crystal form I,a three-wavelength MAD data set was collected at X29B in NSLS,Brookhaven National Laboratory (Upton,NY).Data were indexed,pro-cessed,and merged using HKL2000(Otwinowski and Minor,1997).Twenty-seven out of thirty-six Selenium sites were found and refined using SOLVE(Terwilliger and Berendzen,1999).A native data set to 2.5A˚was used for subsequent manual building and refinement,which was done using standard protocols in phenix.refine and COOT (Afonine et al.,2005;Emsley and Cowtan,2004).Complete data sets of crystal forms II and III were collected at the X25beamline in NSLS.Data were collected at a wavelength corresponding to the Zn 2+-absorption edge (Table 1),as measured using fluorescence scans.All final models have more than 92%of the residues within the most favored regions in the Ramachandran plot and none in disallowed regions.Figures depicting molecular structures were generated using PyMol (DeLano,2002).Endonuclease and DNA-Binding AssaysBsMutL nicking activity was assayed as previously described (Kadyrov et al.,2006),with minor modifications described in the Supplemental Information .To assess DNA binding by BsMutL,supercoiled DNA (5nM)was incubated with BsMutL variants (100nM,dimer)in endonuclease buffer for 90min at 37 C.Reaction mixtures (20m L)were resolved on 1%TAE agarose gels and quanti-fied using ImageJ (/ij/).Data were presented as the mean of three independent measurements,and the error bars correspond to the standard errors of the mean (SEM =s /O n,where s is the average and n the sample size).Zinc-Affinity Fluorescence AssayWild-type and variants of BsMutL-CTD (2m M)were incubated with 1m M Fluo-Zin-3(Invitrogen)in 10mM HEPES (pH 6)and 200mM KCl buffer treated with 1%Chelex-100(Fluka).A calibration fluorescence curve was generated using buffer including increasing concentrations of ZnCl 2(0.25–3m M)in the absence of protein.Spectra (500–600nm)were recorded at an excitation wavelength of 494nm (corrected for buffer effects).Innerfilter effects were neglected because a linear fluorescence intensity response up to stoichiometric amountsMolecular CellStructure of the Endonuclease Domain of MutL150Molecular Cell 39,145–151,July 9,2010ª2010Elsevier Inc.。
