DNA REPLICATION, MUTATION AND REPAIRWang Di （王迪）Ph.D2010-09-08Institute of Immunology, diwang@DNA to RNA to Protein Understanding the central dogma of BiologyDrawn by Ebbe Sloth Andersen.The central dogma states that once “information”has passed into protein it cannot get out again. The transfer of information from nucleic acid to nucleic acid, or from nucleic to protein, may be possible, but transfer from protein to protein, or from protein to nucleic acid, is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein.Francis Crick, 1958DNARNAProteinJames Watson, Francis Crick , Howard Temin, David BaltimoreWhat is DNA?üDNA is deoxyribonucleic acidüIt is a polymer of nucleotidesüIt encodes the information necessary to build a cell üIt allows the storage and replication and execution of cellular information4 types of baseADENINE–THYMINECYTOSINE-GUANINEA T G C DNA contains A, G, C, T, while RNA contains A, G, C, U.DNA StructureNucleic acids arenamed for the typeof sugar: DNA has2'-deoxyribose,whereas RNA hasribose. Thedifference is thatthe sugar in RNAhas an OH groupat the 2' position ofthe pentose ring.DNA: (deoxyribonucleic acid)üdouble helix;übase pairing; A, T, C, Güminor groove and major groove;üsupercoling, open circular and line;üstoring information;üReplicate faithful following base-pairing rules;üMutations: sometime good? sometime bad?DNA is the Genetic Material1.Replicate faithfully2.Have the coding capacity to generate proteins and other products for all cellular functions“A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.”Francis CrickDNA ReplicationüThree models for the replication of DNA üreplication origin 、direction & speed üProteins of DNA ReplicationüDNA synthesis is semidiscontinuousüThe process of DNA replication of E.coli ücircular DNA replicationülinear DNA replicationThree models for the replication of DNAThe hypothesis of semi-conservative replication proposed by Watson and Crick in 1953Meselson-Stahl ExperimentMeselson & Stahl, 1958Replication origin, Direction & Speed Replicon:The unit of DNA in which an individual act of replication occurs is called the replicon. Replication fork: The point at which separation of the strands and synthesis of new DNA takes place.Each replicon "fires" once and only once in each cell cycle.ProkaryoticSingle replicon,single termination,bidirection, tworeplication forksEukaryoticMany replicons andtermination sites,bidirection, manyreplication forks,adjacent replicationbubbles fuse whenthey meet each other.Electron Microscopy of replicating DNA reveals replicating bubbles.Speed of ReplicationProteins of DNA Replication1. DNA Helicases –These proteins bind to thedouble stranded DNA andstimulate the separation ofthe two strands.2. DNA Topoisomerase –This enzyme catalyzes the formation of negative supercoils that is thought to aid with the unwinding process. (Type I and Type II)2. DNA Topoisomerase –DNA topoisomerase IIDNA topoisomerase II3. DNA single-stranded binding proteins （SSB）Stabilizing the single-stranded structure that is generated by the action of the helicases.Replication is 100 times faster when these proteins are attached to the single-stranded DNA.SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteinsSSB Proteins4. Primerase: (DDRP, different from RNA pol) Synthesis a 5-10 nt RNA primer using DNA template for the elongation of lagging strand DNA replication.5. DNA LigaseThe final replication product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3'-hydroxyl and 5'-phosphate groups.DNA Primer synthesis On Lagging strandDNA Ligase5. DNA Polymerase of E.coliArthur Kornberg:He was the first to identify theenzyme catalyzing the synthesis ofDNA, polymerase I. In recognitionof their work elucidating the basicmechanisms of DNA replication,they were awarded the Nobel Prizein 1959.5' to 3' polymerase 3' to 5' exonuclease 5' to 3' exonucleaseDNA Polymerase IThe chain can be cleaved into two parts by proteolytic treatment.The larger cleavage product (68 kD) is called the Klenow fragment .It contains the polymerase and the 3 –5 exonuclease activities.The small fragment (35 kD) possesses a 5 –3 exonucleolytic activity, which excises small groups of nucleotides, up to ~10 bases at a time.DNA Polymerase I Nick TranslationDNA Polymerase III (900KD)ü900 kDü10 proteinsüdifferent types of subcomplexkDa Gene SubassemblySubunitα 130 dnaE | 5'-3'polymerase| POL III COREε 27.5 dnaQ(mutD)θ 10 | 3'-5'exonucleaseτ 71 dnaXγ 47.5 dnaX |δ 35 | ATP dependent δ' 33 | γ COMPLEX clamploaderχ 15 |ψ 12 |β 40.6 dnaN βCLAMP processivityfactorDNA POLYMERASE IIIüTwo copies of the catalytic core. αsubunit (the DNA polymerase activity), εsubunit (3 –5 proofreading exonuclease), and θsubunit (stimulates exonuclease).üTwo copies of the dimerizing subunit, τ, which link the two catalytic cores together.ütwo copies of the clamp, which is responsible for holding catalytic cores on to their template strands.DNA PolymeraseNucleotide polymerizing enzyme, first discovered in 1957DNA Polymerase III Mechanism for PrecisionStructures of DNA polymerase during polymerizing and editing E: exonucleolytic; P: polymerizationDNA replication is semi-discontinuousOkazaki FragmentsSynthesis ofOkazakifragmentsrequires priming,extension,removal of RNA,gap filling, andnick ligation.Structure of the Moving ComplexSummary ICharacters of Circular DNA Replicationθreplication(bacteria)DNA replication with two forks。
