CHAPTER 27: DNA STRUCTURE, REPLICATION, REPAIR LECTURE TOPICS

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 Se...
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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