DNA Replication and Mutation

©2001 Timothy G. Standish

ReplicationReplication


DNA Replication:DNA Replication:How We KnowHow We Know

There are three ways in which DNA could be replicated:

+

NewOld

+

Old

N

ewOld

N

ew

OldConservative - Old double stranded DNA serves as a template for two new strands which then join together, giving two old strands together and two new strands together

OldSemi-conservative - Old strands serve as templates for new strands resulting in double stranded DNA made of both old and new strands

Old

Dispersive - In which sections of the old strands are dispersed in the new strands

+

Old +

N

ewOld +

N

ew

+

Old +

N

ewOld +

N

ew

or


The Meselson-Stahl The Meselson-Stahl ExperimentExperiment

The Meselson-Stahl experiment demonstrated that replication is semiconservative

This experiment took advantage of the fact that nucleotide bases contain nitrogen

Thus DNA contains nitrogen

OH

HOH

P

O

HO ONH2

N N

N N

The most common form of Nitrogen is N14 with 7 protons and 7 neutrons

N15 is called “heavy nitrogen” as it has 8 neutrons thus increasing its mass by 1 atomic mass unit


After 20 min. (1 replication) transfer DNA to centrifuge tube and centrifuge

Disper

sive m

odel

predict

ion

Conservativ

e

model pre

diction

Semi-c

onservativ

e

model pre

diction

The Meselson-Stahl The Meselson-Stahl ExperimentExperiment

Prediction after 2 or more replications

Bacteria grown in N15 media for several replications

Transfer to normal N14 media

X

X

XThe conservative and dispersive models make predictions that do not come true thus, buy deduction, the semi-conservative model must be true.


Tools of ReplicationTools of ReplicationEnzymes are the tools of replication:DNA Polymerase - Matches the correct

nucleotides then joins adjacent nucleotides to each other

Primase - Provides an RNA primer to start polymerization

Ligase - Joins adjacent DNA strands together (fixes “nicks”)


More Tools of ReplicationMore Tools of ReplicationHelicase - Unwinds the DNA and melts itSingle Strand Binding Proteins - Keep

the DNA single stranded after it has been melted by helicase

Gyrase - A topisomerase that Relieves torsional strain in the DNA molecule

Telomerase - Finishes off the ends of DNA strands


Leading StrandLeading Strand

Laging StrandLaging Strand

3’

5’3’

5’

Extension - The Replication ForkExtension - The Replication Fork5’

5’5’3’

3’

5’3’3’

5’

Single strand binding proteins - Prevent DNA from re-anealing

DNA Polymerase

Okazaki fragment

RNA Primers

Primase - Makes RNA primers

5’3’

5’

Gyrase - Relieves torsional strain

Helicase - Melts DNA


Extension - Okazaki FragmentsExtension - Okazaki Fragments

The nick is removed when DNA ligase joins (ligates) the DNA fragments.

3’ 5’5’ 3’

RNA PrimerOkazaki Fragment

RNA and DNA Fragments

Nick

DNA Polymerase has 5’ to 3’ exonuclease activity. When it sees an RNA/DNA hybrid, it chops out the RNA and some DNA in the 5’ to 3’ direction.

DNA Polymerase falls off leaving a nick.

DNAPol.

3’ 5’5’ 3’

RNA Primer

DNAPol.

3’ 5’5’ 3’

RNA PrimerLigase


The Role of DNA GyraseThe Role of DNA Gyrase

Helicase



Helicase

Supercoiled DNA

Gyrase



Gyrase



Gyrase



Gyrase



Gyrase



Gyrase



Gyrase



Gyrase



Gyrase



Gyrase


E. coliE. coli DNA Polymerases DNA Polymerases E. coli has three identified DNA polymerases each of

which has significantly different physical characteristics and roles in the cell

Replication polymerization

10 subunits 600,000 Daltons

II IIIIPolymerase

Major function400 ? 15Molecules/cell

Yes Yes Yes5’- 3’ Polymerization

Yes Yes Yes3’-5’ Exonuclease

Klenow fragment (76,000 Daltons), prepared by mild proteolysis, lacks 5’ to 3’ exonuclease activity and is

used in sequencing

Repair of damaged

DNA

Yes No No5’-3’ Exonulcease

Proofreading/ Removal of

RNA primers109,000 Daltons


Telomere

TelomeraseTelomeraseAt the end of linear chromosomes the lagging strand can’t be completed as the last primer is removed and no 3’ hydroxyl group is available for DNA polymerase to extend from

3’5’5’3’

+

3’5’5’3’

Degradation of RNA primer at the 5’ end

3’5’

5’3’

3’5’5’3’

Next replication


AACCCCAAC

TelomeraseTelomerase

RNA

TelomeraseTelomeraseTelomerase is a ribo-protein complex that adds nucleotides to the end of chromosomes thus restoring their length

GGGTTG5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG


AACCCCAAC


RNA


5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG

GGGTTGGGGTTG


AACCCCAAC


RNA


5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG

GGGTTG GGGTTGGGGTTG


TelomeraseTelomeraseThe TTGGGG repeating telomere sequence can form a hairpin due to unusual GG base pairing

5’GACCGAGCCTCTTGGGTTGGGGTTGGGGTTGGGGTTG3’CTGGCTCGG

O

N

HNH

H

N

N

N

Guanine

O

N

HN H

H

N

N

NGuanine



5’GACCGAGCCTCTTGGGTTGGGGTTGGGG3’GTTGGGG3’CTGGCTCGG

TTGGGGTTGDNA

Pol.



5’GACCGAGCCTCTTGGGTTGGGGTTGGGGAGAACCCAACCCGTTGGGG3’CTGGCTCGG

TT

DNAPol.

Endo-nuclease



5’GACCGAGCCTCTTGGGTTGGG3’CTGGCTCGG

Endo-nuclease

AGAACCCAACCCGTTGGGGT

T

GTTGGGG



MutationMutationWhen Mistakes Are MadeWhen Mistakes Are Made

5’ 3’

5’

DNAPol.

5’

5’ 3’

5’ 3’

5’

DNAPol.

DNAPol.

Mism

atch

3’ to 5’ Exonuclease activity


Thim

ine

Dimer

MutationMutationExcision RepairExcision Repair

3’

5’ 3’

5’

5’ 3’

3’ 5’

Endo-Nuclease


5’ 3’

3’ 5’

5’ 3’

3’ 5’


3’

5’ 3’

5’

Endo-Nuclease

NicksDNAPol.


5’ 3’

3’ 5’


3’

5’ 3’

5’

5’ 3’

3’ 5’

DNAPol.

Endo-Nuclease


5’ 3’

3’ 5’

5’ 3’

3’ 5’


3’

5’ 3’

5’

DNAPol.

Ligase

Endo-Nuclease

Nicks

Nick

Ligase


Break


The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model

Meiosis Prophase I

Homologous chromosomes



Double strand break

Exo-nuclease



Exo-nuclease



Exo-nuclease



Exo-nuclease



DNAPolymerase



DNAPolymerase



DNAPolymerase



DNAPolymerase




Holliday StructureHolliday Structure



Bend



Bend

Twist



Cut



Cut



Cut



Cut




Cutting The Holliday StructureCutting The Holliday Structure

Health & Medicine

DNA Replication and Mutation