Transformation-Griffith’s Expt 1928

DNA Mediates Transformation Convert IIR to IIIS By DNA?

Avery MacLeod and McCarty ExperimentCirca 1943

Transforming Principle

+ means that activity is present DNAse activity All RNA gets degraded during enzyme preparation

A-DNA, B-DNA and Z-DNA The 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 Semiconservative  Each strand serves as a template  Complementary base pairing determines the sequence of the new strand  Each strand of the parental helix is conserved

Possible Modes of DNA 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. coli Note: OriC is 245bp The Core Origin of Replication in SV 40

Prepriming at oriC in E. coli

DNA Polymerases and DNA Synthesis In Vitro

Requirements of DNA Polymerases  Primer DNA with free 3'-OH  Template DNA to specify the sequence of the new strand  Substrates: dNTPs  Mg 2+ ( where?) Nucleophilic attack of alpha phosphate which releases pyrophosphate



 Mg 2+ ( where?)

  

DNA Polymerase I: 5'  3' Polymerase Activity Often called: Kornberg Polymerase

DNA Polymerase I: 5'  3' Exonuclease Activity Cleaves ahead of itself

DNA Polymerase I: 3'  5' Exonuclease Activity Proofreading

Klenow fragment…..is?

DNA Polymerases  Polymerases in E. coli –DNA Replication: DNA Polymerases III and I –DNA Repair: DNA Polymerases II, IV, and V  Polymerases in Eukaryotes –Replication of Nuclear DNA: Polymerase  and/or  –Replication of Mitochondrial DNA: Polymerase  –DNA Repair: Polymerases  and   All 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. coli Note: OriC is 245bp

Prepriming at oriC in E. coli

DNA Replication  Synthesis 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.

What’s 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? GAATTC CTTAAG G -OH p- AATTC CTTAA -p HO- G Cut with EcoR1 Aside:

Calf Intestinal Phosphotase? G -OH p- AATTC CTTAA -p HO- G Cut with EcoR1 G -OH HO- AATTC CTTAA -OH HO- G

Calf Intestinal Phosphotase? p- AATTCgatacagagagactcatgacgG -OH HO- GctatgtctctctgagtactgcCTTAA -p Cut with EcoR1 G -OH HO- AATTC CTTAA -OH HO- G Vector won’t 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 Eukaryotes  Shorter RNA primers and Okazaki fragments  DNA replication only during S phase  Multiple origins of replication  Telomeres

Bidirectional Replication from Multiple Origins in Eukaryotes

The Eukaryotic Replisome

Eukaryotic Replication Proteins  DNA polymerase  -DNA primase—initiation; priming of Okazaki fragments  DNA polymerase  — processive DNA synthesis  DNA polymerase  — DNA replication and repair in vivo  PCNA (proliferating cell nuclear antigen)—sliding clamp  Replication factor-C Rf- C)—loading of PCNA  Ribonuclease H1 and Ribonuclease FEN-1— removal of RNA primers

The E. coli Replisome

The Telomere Problem

Telomerase

Telomere Length and Aging  Most 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 Progression =10 3 =2 10 ….10 more doublings is another 2 10 So 20 doublings is 2 20 = =10 6 So 30 doublings is 2 30 = =10 9 So 40 doublings is 2 40 = =10 12

Molecular Weight of Nucleosides s Base plus ribose Single phosphate 330 Da= 330g/mol/nt (nucleotide) 660 Da= 660g/mol/bp (base pair)

Molecular Weight of Plasmid DNA 330 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 10 3 x 2 x 10 3 = 2 x 10 6 g/mol for a 3kb plasmid 2 x 10 6 g/mol is how many grams per molecule 6 x molecules/mol Thus 2 x 10 6 / 6 x = g/molecules 1g/ 3 x molecules for a given 3kb plasmid

2 x 10 6 g/mol is how many grams per molecule 6 x molecules/mol Thus 2 x 10 6 / 6 x = g/molecules 1g/ 3 x molecules for a given 3kb plasmid 1g/ 3 x molecules is the same as 1mg/ 3 x molecules 1ug/ 3 x molecules 1ng/ 3 x 10 8 molecules 1pg/ 3x 10 5 molecules 1fg/ 3 x 10 2 (300) molecules If each bacterium can hold 3000 molecules, then each Bacterium makes 10fg of plasmid DNA If one makes 1mg of plasmid DNA, then this is fg as well

If each bacterium can hold 3000 molecules, then each Bacterium makes 10fg of plasmid DNA If one makes 1mg of plasmid DNA, then this is fg as well Since each bacterium has 10fg DNA, then only Are needed to produce 1mg DNA. …..So 40 doublings is 2 40 = = cell divisions per hour or about 12 hours 2 36 =~ doublings for bacteria What are the factors that affect DNA replication?

Geometric Doubling Progression =10 3 =2 10 ….10 more doublings is another 2 10 So 20 doublings is 2 20 = =10 6 So 30 doublings is 2 30 = =10 9 So 40 doublings is 2 40 = =10 12