分子生物学的主要脉络：中心法则 课程目标：掌握主要脉络、熟悉基本概念、了解细枝末节,基因的物质状态：DNA上的一个片段 DNA本身的结构（为什么可以作为遗传物质） DNA在细胞中的存在形式（染色质、染色体、基因组的特点） DNA的传代方式：复制 DNA在传代及储存过程中可能遇到的问题及其解决方式 DNA并非静态存储： 同源重组是修复方式和减数分裂之必须； CSSR与转座是不同DNA间的剪切融合-DNA分子大尺度改变的基础 基因表达：DNA序列影响细胞结构功能 RNA的合成：合成工具、基本过程、原核真核异同、后处理 蛋白质的合成：密码、合成工具、基本过程、原核真核异同 基因表达的调控：基本的原则和方法、原核真核异同 基因研究技术,问题： DNA作为遗传物质如何随着细胞分裂而传代？在原核与真核之间的区别与联系？,DNA replication,DNA polymerization Genome replication,1.The Chemistry of DNA Synthesis-化学反应 2. The Mechanism of DNA Polymerase-催化反应的主要酶 3. The Replication Fork: the enzymes-催化反应的其他蛋白,1. The Specialization of DNA Polymerases -细胞内的聚合酶多种用途 2. How DNA pol III work in E. Coli: the trombone model-原核的POL III 3. Initiation of DNA Replication-复制的起始（原核与真核） 4. Finishing replication: Action of Telomerase-复制的终止（原核与真核）,Annealed primer,The Chemistry of DNA Synthesis,template,G = - 3.5kcal/mol,G = - 7kcal/mol,The Mechanism of DNA Polymerase,Palm: for addition of dNTPs and for removal of mispaired dNTP. (1) Binds to two metal ions - alter the chemical environment - catalysis. (2) H-bonds with minor groove of DNA - Monitors the accuracy of the new base-pair,Finger: Binds to the new dNTP; Bends the template; Stabilize the pyrophosphate,Thumb: Holds the primer-template junction in active site,reduces rate of dissociation,template strand,primer strand,dNTP,Nucleotide addition: catalyzed at the active site of DNA polymerase,H- bond contacts between minor groove (new BP) and palm,Two metal ions are held in place by interactions with two highly conserved aspartate residues.,Nucleotide addition: catalyzed at the active site of DNA polymerase,H- bond contacts between minor groove (new BP) and palm,Two metal ions are held in place by interactions with two highly conserved Asp. Metal ion A interacts with the 3-OH reduce the O and H association leaves a nucleophilic 3 O-. Metal ion B interacts with TPs of the new dNTP to neutralize their negative charge. After catalysis, the PPi is stabilized by interacting with metal ion B,Nucleotide addition: catalyzed at the active site of DNA polymerase,H- bond contacts between minor groove (new BP) and palm,Finger domain binds to the incoming dNTP and bends the template,Finger domain binds to the incoming dNTP and bends the template,Finger domain binds to the incoming dNTP and bends the template,Move to enclose dNTP,Finger domain binds to the incoming dNTP and bends the template,DNA polymerase “grips” the template and the incoming dNTP when a correct base pair is made,metal ions,The critical tyr,lys,arg,The base and deoxyribose of the dNTP,The primer,Template strand,Phosphates,Steric constraints空间位阻prevents DNA pol from using rNTP precursors.,How to avoid incorporation of wrong nucleotides,wrong base pair lowers rate of catalysis (kinetic selectivity) 准-稳-快-接； 错-晃-慢-掉,Each bases has its preferred tautomeric form,The occasional flicking of the bases into “wrong” tautomeric form: 10-5 mistake,Exonucleases proofread newly synthesized DNA,The average number of nucleotides added each time the enzyme binds a primer-template junction,The processivity of DNA Polymerase,varies from a few to 50,000 nucleotides,Replication fork: The junction between the newly separated template strands and the unreplicated duplex DNA,Proteins needed at the replication fork,DNA helicase Single strand DNA binding protein (SSB) Primase: to synthesize a short RNA DNA polymerase DNA topoisomerase,DNA helicases unwind double helix in advance of the replication fork,High processivity because they encircle the DNA,This DNA helicase has a 5-3 polarity.,Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism, Cell. 1999 Apr 2;97(1):75-84,Single-stranded binding proteins (SSBs) stabilize single-stranded DNA,Sequence-independent: electrostatic and stacking interactions Cooperative binding: they bind to DNA, also bind to each other,RNA primers must be removed: by RNase H, DNA polymerase & DNA ligase,Topoisomerase removes supercoils produced by DNA unwinding at the replication fork,DNA Pol III holoenzyme: a protein complex responsible for E. coli genome replication,DNA Pols are specialized for different roles in cell,The combined DNA Pol /primase product is between 50100 bp, DNA Polsubstitutes DNA Pol /primase on the leading-strand template and DNA Pol substitutes on the lagging-strand template. and the further extension by them is 10010,000 nucleotides.,DNA polymerase switching during eukaryotic DNA replication,DNA replication,DNA polymerization Genome replication,1.The Chemistry of DNA Synthesis-化学反应 2. The Mechanism of DNA Polymerase-催化反应的主要酶 3. The Replication Fork: the enzymes-催化反应的其他蛋白,1. The Specialization of DNA Polymerases -细胞内的聚合酶多种用途 2. How DNA pol III work in E. Coli: the trombone model-原核的POL III 3. Initiation of DNA Replication-复制的起始（原核与真核） 4. Finishing replication: Action of Telomerase-复制的终止（原核与真核）,DNA Pols are specialized f