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