©2001 Timothy G. Standish Matthew 5:18 18For verily I say unto you, Till heaven and earth pass, one jot or one tittle shall in no wise pass from the law,

©2001 Timothy G. Standish

Matthew 5:18

18 For verily I say unto you, Till heaven and earth pass, one jot or one tittle shall in no wise pass from the law, till all be fulfilled.


Repair:Repair:Maintaining theMaintaining theintegrity of DNAintegrity of DNA

Timothy G. Standish, Ph. D.


DNA ModificationDNA Modification Maintaining DNA integrity is vital to its function A number of mechanisms exist to ensure that the sequence of

nucleotides is maintained in DNA Some of these mechanisms involve the chemical modification

of DNA after replication The most common modification is methylation, in which a

methyl group is added to bases on DNA Methylation functions in:

– Distinguishing between a cell’s DNA and foreign DNA– Distinguishing between old and new DNA strands– Controlling Gene Expression


MethylationMethylation 5-Methylcytosine is the most commonly methylated

nulceotide in E. coli.

61

2

34

N O

NH2

ON

NH2

NCytidine

5

N O

NH2

N O

CH3

N

5-MethylcytidineMethylation

NH2

4-Methylcytosine is less common, but is also known.


N

N

Adenine

N

N

NH2

MethylationMethylation 6-Methyladenine is another common methylated

nulceotide.

1

34

76

28

9

5Methylation N

N

N6-Methyladenine

N

N

NHH3C


E. coliE. coli Methylation Systems Methylation Systems Three methylation systems are known in E. coli:

1 dcm system - Methylates cytosine - Function is unknown

2 dam system - Methylates adenine - Functions in distinguishing new strands of DNA, is involved in control of replication, marks DNA strands for repair and influences transposon activity

3 hsd system - Methylates adenine (cytosine in some bacteria) - Creates specific methylation patterns marking a bacteria’s own DNA and distinguishing it from other species or pathogens’ DNA


Destroying Foreign DNADestroying Foreign DNA Methylase enzymes methylate specific bases in specific sequences of

DNA Only the cells’ own DNA is methylated at a given sequence Thus it is possible to differentiate between the cells’ DNA and DNA that

has been introduced into a cell by a virus or from some other source In bacteria, restriction enzymes are paired with methylases that

recognize the same sequences Restriction enzymes will not cut methylated DNA Thus restriction endonucleases cut up foreign DNA, but not the cell’s

DNA Working with methylases, REs restrict bacteriophages to only one host

bacterial strain.

©2001 Timothy G. StandishLysis

Bacteriophage AttackBacteriophage AttackDestruction of the bacteria’s DNA

Replication of the viral genome

Production of viral parts

Packaging

Infection


Repelling Bacteriophage Repelling Bacteriophage AttackAttack

Methylation sites

M

Methylase


Methylation sites


Unmethylatedmethylation

sites

R Munch! Munch! Munch . . .



Methylation sites

Take that you wicked virus!



Take that you wicked virus!

Methylase and restriction endonucleases must recognize the same sequences if they are to function as an effective system


Restriction EndonucleasesRestriction Endonucleases There are a number of different subclasses of restriction

endonucleasesType I - Recognize specific sequences and cut DNA a nonspecific

site > than 1,000 bp awayType II - Recognize palindromic sequences and cut within the

palindromeType III - Recognize specific 5-7 bp sequences and cut 24-27 bp

downstream of the site. Type II restriction endonucleases are the most useful class as

they recognize specific palindromic sequences in DNA and cut the sugar phosphate backbone within the palindrome


What is a Palindrome?What is a Palindrome? A palindrome is anything that reads the same forwards and

backwards: English palindromes: Mom Dad Tarzan raized Desi Arnaz rat. Able was I ere I saw Elba (supposedly said by Napoleon) Doc note I dissent, a fast never prevents a fatness, I diet on

cod.


DNA PalindromesDNA Palindromes Because DNA is double stranded and the strands run antiparallel,

palindromes are defined as any double-stranded DNA in which reading 5’ to 3’ both are the same

Some examples: The EcoRI cutting site:– 5'-GAATTC-3'– 3'-CTTAAG-5'

The HindIII cutting site:– 5'-AAGCTT-3'– 3'-TTCGAA-5'


Uses of Type II Restriction Uses of Type II Restriction EndonucleasesEndonucleases

Because restriction endonucleases cut specific sequences they can be used to make “DNA fingerprints” of different samples of DNA. As long as the cutting site changes on the DNA or the distance between cutting sites changes, fragments of different sizes will be made.

Because Type II restriction endonucleases cut at palindromes, they may leave “sticky ends” that will base pair with any other fragment of DNA cut with the same enzyme. This is useful in cloning.


G

CTTAA

AATTC

G

1 Digestion

2 Annealing of sticky ends

3 Ligation

Ligase

G

CTTAA

AATTC

G

EcoRIEcoRI

R. E.s and DNA Ligase R. E.s and DNA Ligase Can be used to make recombinant DNACan be used to make recombinant DNA

GAATTC

CTTAAG

GAATTC

CTTAAG

G

CTTAA

AATTC

G

4 Recombinant DNA


QuestionQuestion Where did Type II restriction endonucleases and their

associated methylases come from? In bacteria, restriction enzymes would be lethal in the absence

of the methylase that methylates their recognition site Methylation of specific recognition sites would be pointless in

the absence of restriction enzymes Modification and restriction systems appear to be irreducibly

complex Restriction enzymes and their associated methylase do not

have significant sequence homology, thus they do not share the same DNA recognition domain with different enzyme domains and must have evolved independently


Mutation And RepairMutation And Repair Maintaining the integrity of genetic material is vital to

the survival of organisms Somatic cell mutations are known to lead to cancers in

multicelled eukaryotes Mutations in gametes are passed to offspring and most

commonly will result in decreased fitness Elaborate systems for prevention and repair of

mutations are known in prokaryotes and are believed to exist in eukaryotes although, in eukaryotes, these systems have not yet been well characterized


MutationsMutations Mutation = A random change in the genetic material of a cell Two major types of mutations:

1 Macromutations:– Chromosome number mutations

– Addition or deletion of large chunks of DNA

– Movement of large chunks of DNA

2 Point mutations:– Changes in only one or two bases in a gene

Not all mutations result in phenotypic change


Micro or Point MutationsMicro or Point Mutations Two major types of Micromutations are recognized:

1 Frame Shift - Loss or addition of one or two nucleotides

2 Substitutions - Replacement of one nucleotide by another one. There are a number of different types:– Transition - Substitution of one purine for another purine, or

one pyrimidine for another pyrimidine (more common)– Transversion - Replacement of a purine with a pyrimidine

or vice versa (less common)


Frame Shift MutationsFrame Shift Mutations

5’AGUC-AUG-ACU-UUG-GUA-GUU-GAC-UAG-AAA3’

3’AGTTCAG-TAC-TGA-AAC-CAT-CAA-CTG-ATCATC5’

3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’

5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’

Met Thr Cys Gly Ser

Met Thr ValVal ValLeu

Frame shift mutations tend to have a dramatic effect on proteins as all codons downstream from the mutation are changed and thus code for different amino acids. As a result of the frame shift, the length of the polypeptide may also be changed as a stop codon will probably come at a different spot than the original stop codon.


Purine to Pyrimidine

Transversion

Pyrimidine to Pyrimidine

Transition

Substitution MutationsSubstitution Mutations

3’AGTTCAG-TAC-TGA-ATA-CCA-TCA-ACT-GATCATC5’



Met Thr Cys Gly Ser

3’AGTTCAG-TAC-TGA-AAA-CCA-TCA-ACT-GATCATC5’



Met Thr Cys Gly Ser

5’AGUC-AUG-ACU-UAU-GGU-AGU-UGA-CUAGAAA3’

Met Thr Gly SerTyr

5’AGUC-AUG-ACU-UUU-GGU-AGU-UGA-CUAGAAA3’

Met Thr Gly SerPhe


Transitions Vs TransversionsTransitions Vs Transversions Cells have many different mechanisms for preventing

mutations These mechanisms make mutations very uncommon Even when point mutations occur in the DNA, there may

be no change in the protein coded for Because of the way these mechanisms work, transversions

are less likely than transitions Tranversions tend to cause greater change in proteins than

transitions

ValMutant -globin

H2NOH

OH

CO

H2CH

CCH2

C

O Acid

GluNormal -globin

TC T

Normal -globin DNA

H2NOH

CO

H3CH

CCH

CH3

Neutral Non-polar

AG AmRNA

TC A

Mutant -globin DNA

AG UmRNA

The Sickle Cell Anemia MutationThe Sickle Cell Anemia Mutation



Weakness

Tower skull

Impairedmental function

Infectionsespecially

pneumoniaParalysis Kidney

failureRheumatism

Sickle Cell Anemia:Sickle Cell Anemia:A Pleiotropic TraitA Pleiotropic TraitMutation of base 2 in globin codon 6 from A to T

causing a change in meaning from Glutamate to Valine

Mutant globin is produced

Red blood cells sickle

Heart failure

Pain andFever

Braindamage

Damage to other organs

Spleen damage

Anemia

Accumulation of sickledcells in the spleen

Clogging of smallblood vessels

Breakdown ofred blood cells


Repair SystemsRepair Systems Direct repair - Uncommon: Direct reversal or removal of damage Excision repair - Common: Recognition of damage followed by

cutting out of damaged strand and replacement with a new strand Mismatch repair - Detection of mismatched bases followed by

excision and replacement of one, generally the one on the new strand

Tolerance systems - Important in higher eukaryotes: Used when DNA is damaged so that replication cannot proceed normally. May involve many errors

Retrieval systems - Important in prokaryotes “Recombination repair” damaged sections of DNA are filled in using recombination


Direct RepairDirect Repair The best characterized system of direct repair is

widespread and found in everything from plants to E. coli

DNA strongly absorbs ultraviolet light; this energy may be dissipated by joining adjacent pyrimidines (i.e., thymine) together to form pyrimidine dimers

Photoreactivation of pyrimidine dimers is achieved by the detection of dimers by a light-dependent enzyme that then uses light energy to reverse the reaction and separate the pyrimidines

In E. coli a single enzyme, photolyase (the phr gene product), is responsible for this process

Thymine DimersThymine Dimers

Thymine

Thymine

H

P

O

HO

O

O

CH2

OH

H

P

O

OH

HO

O

O

CH2

O

O

H

H

P OH

O

O

CH2

O

O

H

H OH

P

O

OH

O

O

CH2

NH2

N

N

N

CH 3

O

O

HNN

N

NH2

N

N

N

CH 3

O

O

HNN

N

UVUV LightLight

UVUV LightLight


Thymine

Thymine

OHH

P

O

HO

O

O

CH2

OH

H

P

O

OH

HO

O

O

CH2

O

H OH

O

OCH2

NH2

N

N

N

N

NH2

N

N

N

CH 3

O

O

HNN

N

O

H

H

P

O

O

CH2

O

O

H

P OHOCH 3

O

O

HNN

Ph

otolyaseP

hotolyase

LightLightLightLight


Thymine

Thymine

H

P

O

HO

O

O

CH2

OH

H

P

O

OH

HO

O

O

CH2

O

O

H

H

P OH

O

O

CH2

O

O

H

H OH

P

O

OH

O

O

CH2

NH2

N

N

N

CH 3

O

O

HNN

N

NH2

N

N

N

CH 3

O

O

HNN

N

Ph

otolyaseP

hotolyase


Thymine

Thymine

H

P

O

HO

O

O

CH2

OH

H

P

O

OH

HO

O

O

CH2

O

O

H

H

P OH

O

O

CH2

O

O

H

H OH

P

O

OH

O

O

CH2

NH2

N

N

N

CH 3

O

O

HNN

N

NH2

N

N

N

CH 3

O

O

HNN

N


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

5’ 3’

3’ 5’

MutationMutationExcision RepairExcision Repair

3’

5’ 3’

5’

5’ 3’

3’ 5’

DNAPol.

DNAPol.

Ligase

Endo-Nuclease

Ligase

Nicks



MacromutationsMacromutations Four major types of Macromutations are

recognized:

1 Deletions - Loss of chromosome sections

2 Duplications - Duplication of chromosome sections

3 Inversions - Flipping of parts of chromosomes

4 Translocations - Movement of one part of a chromosome to another part


Macromutation - DeletionMacromutation - Deletion

ChromosomeCentromere

A B C D E F G H

Genes

E F

A B C D G H


Macromutation - DuplicationMacromutation - Duplication

A B C D E F E F G H

ChromosomeCentromere

A B C D E F G H

Genes

E F

Duplication


Macromutation - InversionMacromutation - InversionChromosome

Centromere

A B C D F E G H

Genes

A B C D E F G H

Inversion


Macromutation - TranslocationMacromutation - Translocation

A B E F C D G H

ChromosomeCentromere Genes

A B C D E F G H

Documents

©2001 Timothy G. Standish Matthew 5:18 18For verily I say unto you, Till heaven and earth pass, one jot or one tittle shall in no wise pass from the law,