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Transformation-Griffith’s Expt. 1928. DNA Mediates Transformation. Convert IIR to IIIS By DNA?. Avery MacLeod and McCarty Experiment. Circa 1943. Transforming Principle. DNAse activity. + means that activity is present. All RNA gets degraded during enzyme preparation. - PowerPoint PPT Presentation
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Transformation-Griffiths Expt1928
DNA Mediates TransformationConvert IIR to IIIS By DNA?
Avery MacLeod and McCarty ExperimentCirca 1943
Transforming Principle
+ means that activity is presentDNAse activityAll RNA gets degraded during enzyme preparation
A-DNA, B-DNA and Z-DNAThe Z-DNA helix is left-handed and has a structure that repeats every 2 base pairs. The major and minor grooves, unlike A- and B-DNA, show little difference in width
Non-B DNA in disease
Chapter 10
Replication of DNA and Chromosomes
DNA Replication is SemiconservativeEach strand serves as a templateComplementary base pairing determines the sequence of the new strandEach strand of the parental helix is conserved
Possible Modes ofDNA Replication
The Meselson-Stahl Experiment:DNA Replication in E. coli is Semiconservative
Visualization of Replication in E. coli
Replication in E. coli
The Origin of Replication in E. coliNote:OriC is 245bpThe Core Origin of Replication in SV 40
Prepriming at oriC in E. coli
DNA Polymerases and DNA Synthesis In Vitro
Requirements of DNA PolymerasesPrimer DNA with free 3'-OHTemplate DNA to specify the sequence of the new strandSubstrates: dNTPsMg2+ (where?) Nucleophilic attack of alpha phosphate whichreleases pyrophosphate
Mg2+ (where?)
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DNA Polymerase I:5'3' Polymerase ActivityOften called: Kornberg Polymerase
DNA Polymerase I:5'3' Exonuclease ActivityCleaves ahead of itself
DNA Polymerase I:3'5' Exonuclease ActivityProofreading
Klenow fragment..is?
DNA PolymerasesPolymerases in E. coliDNA Replication: DNA Polymerases III and IDNA Repair: DNA Polymerases II, IV, and VPolymerases in EukaryotesReplication of Nuclear DNA: Polymerase and/or Replication of Mitochondrial DNA: Polymerase DNA Repair: Polymerases andAll of these enzymes synthesize DNA 5' to 3' and require a free 3'-OH at the end of a primer
DNA Polymerase III is the True DNA Replicase of E. coli
DNA replication is a complex process, requiring the concerted action of a large number of proteins.
E. coli DNA Polymerase III Holoenzyme
Replication in E. coli
The Origin of Replication in E. coliNote:OriC is 245bp
Prepriming at oriC in E. coli
DNA ReplicationSynthesis of the leading strand is continuous.Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together.
Whats wrong with this picture?
RNA Primers are Used to Initiate DNA Synthesis
DNA Helicase Unwinds the Parental Double Helix
DNA Ligase Covalently Closes Nicks in DNA
DNA ligase forms a high energy intermediate that
Calf Intestinal Phosphotase?GAATTCCTTAAGG-OH p-AATTCCTTAA-p HO-GCut with EcoR1Aside:
Calf Intestinal Phosphotase?G-OH p-AATTCCTTAA-p HO-GCut with EcoR1G-OH HO-AATTCCTTAA-OH HO-G
Calf Intestinal Phosphotase?p-AATTCgatacagagagactcatgacgG-OH HO-GctatgtctctctgagtactgcCTTAA-pCut with EcoR1G-OH HO-AATTCCTTAA-OH HO-GVector wont religate,But will take in insert
Single-Strand DNA Binding (SSB) Protein
Supercoiling of Unwound DNA
DNA Topoisomerase I Produces Single-Strand Breaks in DNA
DNA Topoisomerase II Produces Double-Strand Breaks in DNA
The Replication Apparatus in E. coli
The E. coli Replisome
DNA Replication in EukaryotesShorter RNA primers and Okazaki fragmentsDNA replication only during S phaseMultiple origins of replicationTelomeres
Bidirectional Replication from Multiple Origins in Eukaryotes
The Eukaryotic Replisome
Eukaryotic Replication ProteinsDNA polymerase -DNA primaseinitiation; priming of Okazaki fragmentsDNA polymerase processive DNA synthesisDNA polymerase DNA replication and repair in vivoPCNA (proliferating cell nuclear antigen)sliding clampReplication factor-C Rf-C)loading of PCNARibonuclease H1 and Ribonuclease FEN-1removal of RNA primers
The E. coli Replisome
The Telomere Problem
Telomerase
Telomere Length and AgingMost human somatic cells lack telomerase activity.Shorter telomeres are associated with cellular senescence and death.Diseases causing premature aging are associated with short telomeres.
BACs
Geometric Doubling Progression12481632641282565121024=103=210.10 more doublings is another 210So 20 doublings is 220=103+3=106So 30 doublings is 230=103+3+3=109So 40 doublings is 240=103+3+3+3=1012
Molecular Weight of NucleosidessBase plus riboseSingle phosphate330 Da= 330g/mol/nt (nucleotide)660 Da= 660g/mol/bp (base pair)
Molecular Weight of Plasmid DNA330 Da= 330g/mol/nt (nucleotide)660 Da= 660g/mol/bp (base pair)
For 3000bp of DNA (a starting plasmid vector)3000 bp x 660 g/mol/bp= 1000 x 3 x 660 = 1x 103 x 2 x 103 = 2 x 106 g/mol for a 3kb plasmid2 x 106 g/mol is how many grams per molecule6 x 1023 molecules/mol
Thus 2 x 106 / 6 x 1023 = g/molecules
1g/ 3 x 1017 molecules for a given 3kb plasmid
2 x 106 g/mol is how many grams per molecule6 x 1023 molecules/mol
Thus 2 x 106 / 6 x 1023 = g/molecules
1g/ 3 x 1017 molecules for a given 3kb plasmid1g/ 3 x 1017 molecules is the same as1mg/ 3 x 1014 molecules1ug/ 3 x 1011 molecules1ng/ 3 x 108 molecules1pg/ 3x 105 molecules1fg/ 3 x 102 (300) moleculesIf each bacterium can hold 3000 molecules, then eachBacterium makes 10fg of plasmid DNAIf one makes 1mg of plasmid DNA, then this is 1012 fg as well
If each bacterium can hold 3000 molecules, then eachBacterium makes 10fg of plasmid DNAIf one makes 1mg of plasmid DNA, then this is 1012 fg as wellSince each bacterium has 10fg DNA, then only 1011 Are needed to produce 1mg DNA...So 40 doublings is 240=103+3+3+3=10123 cell divisions per hour or about 12 hours236=~101136 doublings for 1011 bacteriaWhat are the factors that affect DNA replication?
Geometric Doubling Progression12481632641282565121024=103=210.10 more doublings is another 210So 20 doublings is 220=103+3=106So 30 doublings is 230=103+3+3=109So 40 doublings is 240=103+3+3+3=1012
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