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?)

bg

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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