Transcript Lecture 13 Biol302 Spring 2011

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
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
Note:
OriC is 245bp
The Origin of
Replication in E. coli
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
Mg2+ (where?)
Nucleophilic attack of alpha phosphate which
releases pyrophosphate



Mg2+ (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
Note:
OriC is 245bp
The Origin of
Replication in E. coli
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
Aside:
Calf Intestinal Phosphotase?
Cut with EcoR1
GAATTC
CTTAAG
G-OH p-AATTC
CTTAA-p HO-G
Calf Intestinal Phosphotase?
Cut with EcoR1
G-OH
CTTAA-p
G-OH
CTTAA-OH
p-AATTC
HO-G
HO-AATTC
HO-G
Calf Intestinal Phosphotase?
Cut with EcoR1
p-AATTCgatacagagagactcatgacgG-OH
HO-GctatgtctctctgagtactgcCTTAA-p
G-OH
CTTAA-OH
HO-AATTC
Vector won’t religate,
But will take in insert
HO-G
Single-Strand DNA Binding
(SSB) Protein
Supercoiling of Unwound DNA
DNA
Topoisomerase I
Produces SingleStrand 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  PCNA (proliferating cell
primase—initiation;
nuclear antigen)—sliding
priming of Okazaki
clamp
fragments
 Replication factor-C Rf DNA polymerase —
C)—loading of PCNA
processive DNA
 Ribonuclease H1 and
synthesis
Ribonuclease FEN-1—
 DNA polymerase —
removal of RNA primers
DNA replication and
repair in vivo
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
1
2
4
8
16
32
64
128
256
512
1024=103=210
….10 more doublings is another 210
So 20 doublings is 220=103+3=106
So 30 doublings is 230=103+3+3=109
So 40 doublings is 240=103+3+3+3=1012
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 103 x 2 x 103
= 2 x 106 g/mol for a 3kb plasmid
2 x 106 g/mol is how many grams per molecule
6 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 molecule
6 x 1023 molecules/mol
Thus 2 x 106 / 6 x 1023 = g/molecules
1g/ 3 x 1017 molecules for a given 3kb plasmid
1g/ 3 x 1017 molecules is the same as
1mg/ 3 x 1014 molecules
1ug/ 3 x 1011 molecules
1ng/ 3 x 108 molecules
1pg/ 3x 105 molecules
1fg/ 3 x 102 (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 1012 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 1012 fg as well
Since each bacterium has 10fg DNA, then only 1011
Are needed to produce 1mg DNA.
…..So 40 doublings is 240=103+3+3+3=1012
236=~1011
36 doublings for 1011 bacteria
3 cell divisions per hour or about 12 hours
What are the factors that affect DNA replication?
Geometric Doubling Progression
1
2
4
8
16
32
64
128
256
512
1024=103=210
….10 more doublings is another 210
So 20 doublings is 220=103+3=106
So 30 doublings is 230=103+3+3=109
So 40 doublings is 240=103+3+3+3=1012