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
