7 013 molbioi 2 26 07 - mitweb.mit.edu/7.01x/7.013/documents/molecularbiolecture1.pdf ·...
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Contact Prof. Sive
Put “7.013 student in
subject line!
Office hours
Mon, Fri after class, outside 10-250
Tue 11-12 Whitehead Inst. 401C
or by arrangement
7.013
2.28.07
Molecular Biology I
(DNA replication)
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How-to 1How-to 1 FO
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How-to 2How-to 2
SYSTEMSSYSTEMS
PR
OB
LE
MS
PR
OB
LE
MS
BIOCHEM GENETICS CELL BIO.MOL. BIO
STEM
CELLS,
CLONING
REC. DNA
CELL TYPE
3DSTRUCTURE
VIRUSES
CANCER
HUMAN
DISEASE
LIFELIFE
NERVOUSIMMUNE
SYSTEMS
BIOLOGY
FUTUREFUTURE
Review
Base pairing in DNA
H-bond
Each base-
pair: one
purine and
one
pyrimidine;
same overall
shape
1
Purves 11.6:
Base pairing and uniform shape
of base pairs
leads to formation
of the DNA double helix
2
“prime” = #’s on
the d-ribose
Purves 11.7. Antiparallel, complementary DNA strands
3
1. Principles
double-stranded
DNA
5’3’
3’5’
replication of
complementary strands
Replication of identical DNA molecules
strand-separation
templatetemplate
5’3’
3’5’
H. Sive MIT 2007
4
5’3’
3’5’
double-stranded
DNA
Replication of identical DNA molecules
GATTACA
CTAATGT
GATTACA
strand-separation
CTAATGT
templatetemplate
H. Sive MIT 2007
5
replication of
complementary strands
GATTACA CTAATGT
CTAATGT GATTACA
DNA replication animation/ oversimplification!
6
5’3’
3’5’
double-stranded
DNA
strand- separation, replication from each 3’ end
Generation of identical DNA molecules
by replication of complementary strands
5’
5’
3’
3’
H. Sive MIT 2007
7 See Purves 11.10
Purves 11.10: DNA polymerization:
new nucleotides are added to the 3’OH
8
2. Practice
origins of replication
ori ori
9
+H. Sive MIT 2007
Newly replicated DNA
Bidirectional synthesis
Parental ds DNA
Replicated ds DNA
binds specific proteins
which activate
strand separation
and replication
2. Practice
enzymes
5’3’
3’5’
primer removal5’3’
3’5’ H. Sive MIT 2007
10
5’ 3’RNA primer
synthesisPrimase
Only 1 strand
shown
See Purves 11.14
strand-separation Helicase
Single-stranded
DNA binding
protein
3’polymerization
5’ 3’ DNA polymerasePurves 11.13
11
Purves 4.9
A big problem:
how to rapidly replicate >108 bp per chromosome12
2. Practice
continuous/discontinous
DNA synthesis
ori = origin of replication
5’3’
3’5’
ori
= primer
5’
3’
5’
3’
H. Sive MIT 2007
13
3’
5’
leading strand
lagging strand
leading strand te
mplate
lagging strand template
Continuous
DNA synthesis
Discontinous
DNA synthesis
See Purves 11.6
3’
5’
leading strand
lagging strand
Continuous
DNA synthesis
Discontinous
DNA synthesis
H. Sive MIT 2007
See Purves 11.6
Primer removal by RNAase,
DNA fragments act as primers to
“fill in” gaps and ligase
joins fragments
leading strand
lagging strand
3’
5’
14
DNA replication animation
15
3. DNA repair
will be completed on 3.2.07!!
5’3’
3’5’
double-stranded
DNA
Replication of identical DNA molecules
GATTACA
CTAATGT
GATTACA
strand-separation
CTAATGT
templatetemplate
H. Sive MIT 2007
replication of
complementary strands
GATTACA CTAATGT
CTAATGT GATTACA
5’3’
3’
5’
double-stranded
DNAGATTACA
CTAATGT
GATTACA
strand-separation (one strand shown)
template
H. Sive MIT 2007
16
Outcome of
incorrect replication
incorrect replication
round 1
GATTACA 3’3’ CGAATGT
3’
GATTACA 3’CGAATGT3’
strand-separation
replication round 2
GCTTACA 3’3’ CTAATGT
mutant DNA molecule wild type DNA molecule
5’3’
3’
5’
double-stranded
DNAGATTACA
CTAATGT
GATTACA
strand-separation
template
H. Sive MIT 2007
17
replication of
complementary strand
requires “base pairing site” on DNAPol
GATTACA
ATAATGT
incorrect base!!!
5’3’
3’5’
removed by
3’ to 5’ exonuclease
“proofreading site” on DNAPol
GATTACA
CTAATGTDNA polymerase tries again
correct base!!
Proofreading
TAATGT3’
See Purves 11.19
5’3’
3’
5’
double-stranded
DNAGATTACA
CTAATGT
G TTACA
strand-separation
template
H. Sive MIT 2007
18
incorrect base incorporated,
makes a “bubble”, without H bonds
GATTACA 3’
incorrect removed
correct base filled in
GATTACA
Mismatch repair
3’ C AATGT
A
G
3’ C AATGT
CTAATGT
3’
3’3’
How do repair enzymes know
which strand is correct?
Parental “A/C”s are methylated.
MeMeMe
MeMeMe
MeMeMe
MeMeMe
See Purves 11.19
Protective suits for people with Xeroderma
Nucleotide excision repair
of damaged DNA
(Xeroderma defect)
caused by UV light!!
19
See Purves 11.19