chapter 27: dna structure, replication, repair lecture...
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CHAPTER 27: DNA STRUCTURE, REPLICATION, REPAIR
LECTURE TOPICS
1) DNA STRUCTURE Models vs X-ray
Static vs Dynamic
2) DNA-PROTEIN INTERACTIONSSequence-specific vs non-specific
3) DNA TOPOISOMERASES – Changes in state of DNA
Cutting and sealing strands
4) DNA REPLICATION
E. coli chromosomeThe 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
-Structure details are sequence specific (dependent)- sequence provides unique 3-D fit for protein-DNA interactions
Structure is regular and not dependent on base sequence
Base roll (bends DNA)Adjacent base pairs parallel
Propeller twisting (bases)Paired bases in same plane
28-42 turn/base pair36o turn/ base pair
“Real” B-DNA structureWatson-Crick Model
Models vs Real DNA structure from x-ray diffraction
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 Models vs X-ray
Static vs Dynamic
2) DNA-PROTEIN INTERACTIONSSequence-specific vs non-specific
3) DNA TOPOISOMERASES – Changes in state of DNA
Cutting and sealing strands
4) DNA REPLICATION
E. coli chromosomeThe players and the process
5) DNA RECOMBINATION
6) DNA MUTATIONS AND REPAIR
Base pairs: H-bonding properties
D D
D
A
A
A
A
A
A A
A
Bases are H-donors (D) or acceptors (A)
A:T G:C
Up
Down
B-DNA A-DNA (and RNA)
3` 5` phosphodiester bond
RNA 2`OHSteric Hindrance
Z – DNA: exists but Function unknown
DNA-RNA or (RNA-RNA)DNA
Z B A
DNA-RNAMost DNA RARE
RNA-RNA
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!!]
Minor Groove
__
+
+(DNase I)
_
_
_
_
_
Arg / Lys have (+)charge in protein
Non-specific interactions: Deoxyribonuclease I
Sugar – Phospate backbone(-)charge
Major Groove_
EcoRV restriction enzyme recognition site
2-fold symmetry
!! Asymmetrical DNA Recognition Site!![Fig.9-37]
Sequence-specific interactions
EcoRV
GATATC
CTATAG
DNA bases form specific H-bonds with loop of EcoRV protein β-turn
Opens 500
Induced Fit
Sequence specific Interactions in DNA major groove
DNA
EcoRV bends (kinks) DNA by 500
250 250
[Fig.9-40]
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
CTTAAGl l l l l l
• Each DNA strand forms 6 H-bonds with Gluand Arg residues of Eco RI the enzyme.
• A total of 12 H-bonds form in Enzyme-DNA complex.
•
EcoRI - DNA complex
~ Half a helix turn
Top
One side
Two kinds of EcoRI-DNA Interactions
DNA
Protein
(+) dipole-phosphate backbone (-) interactions (at a specific location)
Arg
G base
specific H-bonds
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’ phosphodiesterbond.
• Ligase “Joins” pieces of DNA by making a 3`- 5` phosphodiester bond
Topoisomerases: Change state of DNA supercoiling
[Fig.27-2]
[demonstrate with model]
Topoisomerases: Change state of DNA supercoiling
0 min 5 min 30 min
Add topoisomerase
2 kinds of supercoils
Negative(right-handed)
Positive(left-handed)
Topoisomerases can convert (+) to (-) supercoils
Topoisomerase(s) II
2 strands cutRight-handed supercoils(DNA gyrase uses ATP)
Topoisomerase(s) I
1 strand cutLeft-handed supercoils[Helicase in DNA synthesis makes NO cuts, uses ATP]
Topoisomerase I – cuts one strand
Negative (- 5) Positive (- 4)supercoils
cut
(-) 1 Supercoil changes (+) 1
Topoisomerases II make 2 cuts (Ex: DNA Gyrase)
Cuts 2 strands
Mechanism of DNA Gyrase (a Topoisomerase II)
Left-handed
[“Bad” (Stress)]
+1
-1
5`-P linked to Tyrosine on A subunits
Net –2 linking # Right-handed [“Good”]
l l l l l l l l l l l l l l l l l l l l3`OH 5`P
DNA nick
l l l l l l l l l l l l l l l l l l l l l-P-
DNA Ligase: Makes a 3`- 5` phosphodiester bond
Joins a 3`OH with a free 5`-Phosphate of Adjacent bases
..
Nucleophilic attack
• requires a “nick”(break) in a 3’-5’ phosphodiester bond.
• “Joins” pieces of DNA by making a 3`- 5` phosphodiester bond
DNA LIGASE Reaction
2Pi PPi AMP
New 3`- 5` phosphodiester bond
Summary: DNA TOPOISOMERASES
DNA LIGASE
uses ATPMakes new 3`- 5` bond
TOPOISOMERASE I
Adds (+) supercoilsNo ATP required1-strand cut
HELICASE
Adds (+) supercoilsNeeds ATPNo Cuts
DNA GYRASE
Adds (–) supercoils2 strand cutNeeds ATP
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
TOPIC REVIEW
1) DNA STRUCTURE
Models vs X-ray
Static vs Dynamic
2) DNA-PROTEIN INTERACTIONS
Sequence-specific vs non-specific
3) DNA TOPOISOMERASES – Changes in state of DNA
Cutting and sealing strands
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
LECTURE TOPICS
4) DNA REPLICATION
E. coli chromosomeThe 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` 3` Polymerase)
[Processive enzyme adds 20 bases at 10/sec]
2. Proofreading: 3` 5` Exonuxlease (corrects last error)
3. Error Correcting 5` 3` exonuclease (repairs old errors)
DNA polymerase I (Pol I) reaction mechanism: 5’ to 3’ polymerase [see Ch.5 notes]
3`
5’
Error Rate: 1/10,000 bases (10-4)
5’
Nucleophilicattack
**
New base
Pol I proof reading exonuclease (3’ 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
3’
5’
The “Central Dogma” of molecular biology
DNA RNA PROTEIN
Replication Reverse
DNA virus Retrovirus RNA Virus
Transcription translation
transcription
Prions
10 -4,-5
10-810-3, -4
10-4
FEATURES OF PROCESSES
Accuracy, Signals, Stage
One error in 108 bases polymerized
In E. coli, 4x106 bases x 2 DNA strands ~ 107 bases per replication
This is 1 mistake in 10 cells
mismatches
(Exonuclease)
5` 3`cut
Pol I exonuclease (5’ 3’ editing)removes pre-existing errors
5`
5` 3` 5` 3`
Question: Is Pol I sequence specific??
Pol I Klenow fragment
3` OH
α-Pd
2- Mg2+ metal ions in Pol I active site play a role in
5’ to 3’ polymerase mechanism
Pol I (donor) H-bonds to base pair acceptors
Minor Groove H-bonds
Base pair functional group acceptors*are same for A-T
and G-C base pairs
A T
**
Pol I : Incoming dNTP causes formation of tight binding pocket in 5’ to 3’ polymerase
d
d
Pol I 3` 5` exonuclease(edits a mistake)
Unzip base-paired section
Cut wrong base
Move cut strand to exonuclease site
Leave 3`-OH
Observation :E. coli mutants lacking Pol I replicate DNA and grow normally.
How??DNA POLYMERASES II and III discovered (late 1960’s)
Have 5` to 3` polymerase (like Pol I) and proofreading 3` to 5` exonuclease
No 5` to 3` exonuclease activity
Pol III used for chromosomal DNA replication (processive – 1000 base pairs / second)
Many other proteins also involved in replication
DNA POLYMERASE III (Pol III)
Holoenzyme is an asymmetric dimer
Catalysis
Pol IIIβ2 - dimers
Pol III is processive :
Adds thousands of bases
1000 / 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 POLYMERASE I – Three different activities• Template directed 5` 3` polymerase
• Proofreading (3` 5' exonuclease)
• Error-Correcting (5` 3` Exonuclease)
• E. coli mutants lacking Pol I have normal growth andDNA 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
DNA POLYMERASES: SUMMARY
Ori C : 254 b.p. Origin of Replication
[Start signal for Initiation of replication]
E. Colichromosome
replicating looks like this:
(theta structure)
Replication fork
Replication fork
ELONGATION: Direction of DNA synthesis is 5` 3`
Apparent 3` 5`
Actual 5` 3`(as always)
(Discuss first)
(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` 3` Exonuclease
Pol I 5` 3`synthesis
DNA ligase Okazaki fragmentsjoined
Pol I removes primer
Some DNA replication proteins in E. coli
(+/-) supercoilsadded
•E. Coli chromosome contains 400,000 turns of helix
•Need 100 turns / second
E. Coli replication fork(+) (-) has gyrase too!
(SSB)
5’
Inverted loopPol III dimerholoenzymesynthesizes both strands at fork. 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 minutesNeed > 6000 replication forks!!
Eukaryotic chromosome: Problem at end of replication
[New histones]
[Old histones]
??
One daughter molecule would get
shorter and
shorter!
(telomere)
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
Telomerase
RNA Primer
New DNA
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 mismatch mutation
A:T to G:C
A transition mutation [purine to purine]
CA
*
*
Chemical Mutagen : Nitrous acid (HNO2)
C
Deamination causes A:T to G:C transitions
HNO2 also deaminates C to U: causes G:C to A:T transitions
A “A” C
*
C:A mismatch mutation
Base Analog Mismatch: Thymine analog 5-BU
“T” G
5-BU:T mismatch A to T mutation
Should be ALooks like C
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)
*
* * *
Repair of T-T dimers Remove several bases
1) Base excision repair
2) In place repair (pyrimidine dimers)
3) Excision repair
T-T dimers (adjacent bases on same strand of DNA)
Sunlight:
UV light causes T-Tdimer formation
*
*
Repair of T-T dimersCut Cut
excinuclease
Pol I
Ligase
5`
3`
3`
5`
OH P
T-T
Pol I
C UC4- (NH2) to C4- (C=O)
Remove uracil
Cut 3`-5` Phosphodiester
bond
Pol I + Ligase
Repair of uracil in DNA:[uracil would lead to C to T transition]
* **
T
Mismatch Repair: Occurs soon after a DNA replication error
Template
No CH3- A on new DNA
Up to 2000 bases removed
Synthesize again
Endonuclease
Ligase
New DNA
Exonuclease
Pol III
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 His+ revertantsMutagen
+ liver extract
CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR
SEE KEY CONCEPTS: P.1 ONLINE LECTURE NOTES