Download CHAPTER 27: DNA STRUCTURE, REPLICATION, REPAIR

CHAPTER 27: DNA STRUCTURE, REPLICATION, REPAIR
LECTURE TOPICS
1) DNA STRUCTURE
Æ Models vs X-ray
Æ Static vs Dynamic
2) DNA-PROTEIN INTERACTIONS
Æ Sequence-specific vs non-specific
3) DNA TOPOISOMERASES – Changes in state of DNA
Æ Cutting and sealing strands
4) DNA REPLICATION
Æ E. coli chromosome
Æ The players and the process
5) DNA RECOMBINATION
6) DNA MUTATIONS AND REPAIR
E.coli is:
Mr. DNA replication
Mr. Transcription
Mr. Translation
Mr. Control of gene expression
The Boss
The E. is Escherichia, but you can call me Ed
Models vs Real DNA structure from x-ray diffraction
Watson-Crick Model
“Real” B-DNA structure
36o turn/ base pair
28-42 turn/base pair
Paired bases in same plane
Propeller twisting (bases)
Adjacent base pairs parallel
Base roll (bends DNA)
Structure is regular and not
dependent on base sequence
-Structure details are sequence
specific (dependent)
- sequence provides unique 3-D
fit for protein-DNA interactions
REAL B-DNA from X-ray structure
BENDING OF DNA-B
DNA: Bases are not in a plane
PROPELLER TWISTING
REAL B-DNA from X-ray structure
Show Movie
CHAPTER 27: DNA STRUCTURE, REPLICATION, REPAIR
LECTURE TOPICS
1) DNA STRUCTURE
Æ Models vs X-ray
Æ Static vs Dynamic
2) DNA-PROTEIN INTERACTIONS
Æ Sequence-specific vs non-specific
3) DNA TOPOISOMERASES – Changes in state of DNA
Æ Cutting and sealing strands
4) DNA REPLICATION
Æ E. coli chromosome
Æ The players and the process
5) DNA RECOMBINATION
6) DNA MUTATIONS AND REPAIR
Base pairs: H-bonding properties
D
A
A
A
D
A
A
A
A
A:T
A
D
G:C
Bases are H-donors (D) or acceptors (A)
B-DNA
A-DNA (and RNA)
Up
Down
RNA 2`OH
Steric Hindrance
3`Æ5`
phosphodiester bond
DNA
DNA-RNA
or (RNA-RNA)
Z – DNA: exists but
Function unknown
Z
B
A
DNA-RNA
RNA-RNA
Most DNA
RARE
Table 27-1 frp,, 5th
DNA-Protein Interactions
(DNA and/or RNA)
[A key concept for rest of the course]
- Non-specific [DNA sequence independent]
- Specific
[ DNA Sequence matters!!]
Non-specific interactions: Deoxyribonuclease I
_
Major Groove
_
_
Arg / Lys have (+)
charge in protein
Minor Groove
_
+ _
(DNase I)
+ _
_
_
Sugar – Phospate backbone
(-)charge
EcoRV restriction enzyme recognition site
Sequence-specific interactions
2-fold symmetry
!! Asymmetrical DNA Recognition Site!!
[Fig.9-37]
EcoRV
GATATC
DNA bases form specific H-bonds
with loop of EcoRV protein β-turn
CTATAG
Opens 500
Induced Fit
DNA
Sequence specific Interactions in DNA major groove
EcoRV bends (kinks) DNA by 500
250
[Fig.9-40]
250
EcoRV β-turn loops
H-bond with DNA
Specific H-bonds in each
EcoRV monomer
*
*
*
* *
[Fig.9-39]
* *
*
*
*
*
Evolution: DNA sequence elements are
conserved in active sites of some Type II
restriction enzymes
EcoRI recognition site
GAATTC
•
CTTAAG
l
l
l l l
l
Each DNA strand forms 6 H-bonds with Glu
and Arg residues of Eco RI the enzyme.
•
• A total of 12 H-bonds form in Enzyme-DNA
complex.
EcoRI - DNA complex
One side
~ Half a helix turn
Top
Two kinds of EcoRI-DNA Interactions
DNA
(+) dipole-phosphate backbone (-)
interactions (at a specific location)
Protein
Arg
specific
H-bonds
G base
Circular DNA problem: How are ends of linear
DNA joined to form circular DNA?
Solution: (1967) DNA ligase was discovered
Ligase was first in a NEW CLASS of enzymes
called DNA Topoisomerases. These enzymes
change DNA topology. [demonstrate with model]
•
Ligase requires a “nick”(break) in a 3’-5’ phosphodiester
bond.
•
Ligase “Joins” pieces of DNA by making a 3`- 5`
phosphodiester bond
Topoisomerases: Change state of DNA supercoiling
[demonstrate with model]
[Fig.27-2]
Topoisomerases: Change state of DNA supercoiling
Add topoisomerase
0 min
5 min
30 min
2 kinds of supercoils
Negative
(right-handed)
Positive
(left-handed)
Topoisomerases can convert (+) to (-) supercoils
Topoisomerase(s) II
2 strands cut
Right-handed supercoils
(DNA gyrase uses ATP)
Topoisomerase(s) I
1 strand cut
Left-handed supercoils
[Helicase in DNA
synthesis makes NO
cuts, uses ATP]
Topoisomerase I – cuts one strand
Negative (- 5)
supercoils
Positive (- 4)
cut
Topoisomerases II make 2 cuts (Ex: DNA Gyrase)
Cuts 2 strands
(-) 1
Supercoil changes
(+) 1
Mechanism of DNA Gyrase (a Topoisomerase II)
5`-P linked to
Tyrosine on A
subunits
+1
Left-handed
[“Bad” (Stress)]
-1
Net –2
linking #
Right-handed [“Good”]
DNA Ligase: Makes a 3`- 5` phosphodiester bond
•
•
requires a “nick”(break) in a 3’-5’ phosphodiester bond.
“Joins” pieces of DNA by making a 3`- 5` phosphodiester bond
l l l l l l l l l l l
..
3`OH 5`P
Nucleophilic attack
l l l l l l l l l l l l l
l l l l l l l l l
DNA nick
-P- l l l l l l l l
Joins a 3`OH with a free 5`-Phosphate of
Adjacent bases
DNA LIGASE Reaction
2Pi Å PPi
New 3`- 5` phosphodiester bond
AMP
Summary: DNA TOPOISOMERASES
DNA LIGASE
uses ATP
Makes new 3`- 5` bond
TOPOISOMERASE I
Adds (+) supercoils
No ATP required
1-strand cut
HELICASE
Adds (+) supercoils
Needs ATP
No Cuts
DNA GYRASE
Adds (–) supercoils
2 strand cut
Needs ATP
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
TOPIC REVIEW
1) DNA STRUCTURE
Æ Models vs X-ray
Æ Static vs Dynamic
2) DNA-PROTEIN INTERACTIONS
Æ Sequence-specific vs non-specific
3) DNA TOPOISOMERASES – Changes in state of DNA
Æ Cutting and sealing strands
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
LECTURE TOPICS
4) DNA REPLICATION
Æ E. coli chromosome
Æ The players and the process
5) DNA RECOMBINATION
6) DNA MUTATIONS AND REPAIR
DNA REPLICATION
•
DNA POLYMERASES
•
THE REPLICATION PROCESS
DNA polymerase I (Pol I) has 3 different activities
1. Template- Directed DNA polymerase (5`Æ 3` Polymerase)
[Processive enzyme adds 20 bases at 10/sec]
2. Proofreading: 3`Æ 5` Exonuxlease (corrects last error)
3. Error Correcting 5`Æ3` exonuclease (repairs old errors)
DNA polymerase I (Pol I) reaction mechanism:
5’ to 3’ polymerase [see Ch.5 notes]
5’
Nucleophilic
attack
3`
New base
5’
**
Error Rate: 1/10,000 bases (10-4)
Pol I proof reading exonuclease
(3’ Æ 5’ editing)
• removes wrong base if inserted
(leaves a 3`OH)
Error rate is also 1x10-4
Total Error Rate for Pol I DNA synthesis
and editing = 10-4 x 10-4 = 10-8
5’
3’
The “Central Dogma” of molecular biology
10 -4,-5
10-3, -4
10-8
Transcription
translation
DNA
RNA
Replication
Reverse
transcription
DNA virus
Retrovirus
RNA Virus
PROTEIN
Prions
10-4
FEATURES OF PROCESSES
‰ Accuracy, Signals, Stage
8
‰ One error in 10 bases polymerized
6
7
‰ In E. coli, 4x10 bases x 2 DNA strands ~ 10 bases per replication
‰ This is 1 mistake in 10 cells
Pol I exonuclease (5’ Æ 3’ editing)
removes pre-existing errors
mismatches
(Exonuclease)
5`
5`
cut
3`
Pol I Klenow fragment
5`Æ3`
5`Æ3`
Question: Is Pol I sequence specific??
+
2- Mg2 metal ions in Pol I active site play a role in
5’ to 3’ polymerase mechanism
d
3` OH
α-P
Pol I (donor) H-bonds to base pair acceptors
T
A
*
Minor Groove
H-bonds
*
Base pair functional
group acceptors*
are same for A-T
and G-C base pairs
Pol I : Incoming dNTP causes formation of
tight binding pocket in 5’ to 3’ polymerase
d
d
Pol I 3`Æ 5` exonuclease
(edits a mistake)
Move cut strand to
exonuclease site
Cut wrong base
Leave 3`-OH
Unzip base-paired section
Observation :
E. coli mutants lacking Pol I replicate DNA and grow normally.
How??
DNA POLYMERASES II and III discovered (late 1960’s)
Æ Have 5` to 3` polymerase (like Pol I) and proofreading
3` to 5` exonuclease
Æ No 5` to 3` exonuclease activity
ÆPol III used for chromosomal DNA replication
(processive – 1000 base pairs / second)
Æ Many other proteins also involved in replication
DNA POLYMERASE III (Pol III)
Catalysis
Holoenzyme is an asymmetric dimer
Pol III is processive :
Pol III
β2 - dimers
ÆAdds thousands of bases
Æ1000 / sec (Pol I is 10 / sec)*
* Pol III is 100 times as fast as Pol I
Question: How many minutes to replicate E. coli DNA?
DNA POLYMERASES: SUMMARY
DNA POLYMERASE I – Three different activities
•
•
•
•
Template directed 5`Æ3` polymerase
Proofreading (3`Æ5' exonuclease)
Error-Correcting (5`Æ3` Exonuclease)
E. coli mutants lacking Pol I have normal growth and
DNA replication
DNA POLYMERASES II AND III
•
Have 5` to 3` polymerase and proofreading 3` to 5`
exonuclease
•
•
Pol III replicates the E. coli chromosome
Many other proteins are also involved
Ori C : 254 b.p. Origin of Replication
[Start signal for Initiation of replication]
E. Coli
chromosome
replicating looks
like this:
(theta structure)
Replication fork
Replication fork
ELONGATION: Direction of DNA synthesis is 5`Æ 3`
Apparent 3` Æ 5`
(Discuss first)
Actual 5` Æ 3`
(as always)
(Discuss later)
Helicase unwinds DNA
•
Uses ATP as energy
•
Introduces positive supercoils
Initiation of DNA synthesis
(An RNA primer is extended 5’ – 3’)
(An RNA polymerase)
• Both strands
• Almost all
chromosome
DNA synthesis
Termination of DNA synthesis
Pol I 5`Æ3` Exonuclease
Pol I removes primer
Pol I 5`Æ3`synthesis
DNA ligase
Okazaki fragments
joined
Some DNA replication proteins in E. coli
(+/-) supercoils
added
•E. Coli chromosome contains 400,000
turns of helix
•Need 100 turns / second
E. Coli replication fork
(+)
(SSB)
(-) has gyrase too!
5’
Pol III dimer
holoenzyme
synthesizes
both strands
at fork.
Inverted loop
Primer
Lagging strand
(1,000 bases average length)
Leading strand
Eukaryotic chromosome replication
Elongation is bi-directional from thousands of forks.
Ex : Drosophila chromosome (size – 62 x 106 bp)
Replication rate is 2.6 kb/min/origin.
To replicate the chromosome:
16 days with only one origin
Actual rate : < 3 minutes
Need > 6000 replication forks!!
Eukaryotic chromosome: Problem at end of replication
(telomere)
[New histones]
[Old histones]
??
One
daughter
molecule
would get
shorter
and
shorter!
End of Chromosome termination solution
TELOMERASE
•
•
•
•
A ribonucleoprotein complex (RNA + protein)
A Reverse transcriptase with an RNA template
Processive
Adds 100’s of short repeated sequence to incomplete
3` ends of chromosomes
Telomere
100,s of GGGTTG
added
RNA Primer
New DNA
Telomerase
Many repeats
of new DNA
TELOMERASE
Chromosome
3` end
The end of the telomere (May 1999)
The new view
Telomere: Repeated sequences form base pairs
5’
3’
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
LECTURE TOPIC
5) DNA RECOMBINATION
DNA Recombination: Occurs between
molecules that have similar sequences
Homologous Recombination results in:
• Gene replacement
• Gene disruption
*
*
*
*
* [Shared sequences]
Recombined Gene with some different bases
[Fig.6-31]
“Recombinase” (Cre- a Type I topoisomerase)
*
*
*
*
*
*
*
*
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
LECTURE TOPIC
6) DNA MUTATIONS AND DNA REPAIR
4 SIGNS of MALIGNANT MELANOMA
MUTATIONS ARISE FROM MISMATCHED BASES IN DNA
•
Persistent replication errors are actually only 10-9 to 10-10
[DNA repair improves error from 10-8]
•
•
Chemical mutagens
Ultraviolet light (Sunlight)
DNA REPAIR
•
•
•
Base excision [uracil removal]
T-T dimer removal [defect in Xeroderma pigmentosum]
Mismatch repair [defects in colorectal, stomach, uterine
cancers]
IS A MUTAGENIC AGENT ALSO CARCINOGENIC??
•
Ames test [Reversion of Salmonella His- to His+ phenotype]
A replication error
C
A
*
C:A mismatchÆ mutation
A:T to G:C
A transition mutation [purine to purine]
*
Chemical Mutagen : Nitrous acid (HNO2)
Deamination causes A:T to G:C transitions
A
“A”
C C
C:A mismatchÆ mutation
*
HNO2 also deaminates C to U:
causes G:C to A:T transitions
Base Analog Mismatch: Thymine analog 5-BU
5-BU:T mismatchÆ A to T mutation
“T”
Looks
like C
G
Should be A
Intercalating mutagens fit between adjacent base pairs
• cause base insertions, leading to translational frameshifts
Same size as a base pair
DNA Chemical Adducts
Epoxide
[reacts withN7
of Guanine
and forms
covalent link]
DNA Repair: 3 types
Altered base
(3-CH3-Adenine)
*
*
1) Base excision repair
*
*
2) In place repair
(pyrimidine dimers)
Repair of
T-T dimers
Remove several bases
3) Excision repair
T-T dimers (adjacent bases on same strand of DNA)
*
Sunlight:
UV light causes T-T
dimer formation
*
Repair of T-T dimers
Cut
T-T
Cut
excinuclease
OH
5`
P
Pol I
3`
Pol I
Ligase
5`
3`
C4- (NH2) to C4- (C=O)
C
U
*
*
*
Remove uracil
T
Cut 3`-5`
Phosphodiester
bond
Repair of uracil in DNA:
Pol I + Ligase
[uracil would lead to C to T
transition]
Mismatch Repair: Occurs soon after a DNA replication error
Template
New DNA
No CH3- A on
new DNA
Exonuclease
Endonuclease
Up to 2000
bases removed
Pol III
Synthesize again
Ligase
Triplet repeat expansions in eukaryotic DNA:
(Associated with neurological diseases)
Loop lets red strand get longer
with 3 more repeats added
Ames test: Are mutagens also carcinogens?
Medium lacks histidine
His- mutants
Mutagen
+
liver extract
His+ revertants
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
SEE KEY CONCEPTS: P.1 ONLINE LECTURE NOTES