1. 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.

2. Restriction and Repair: Maintaining the integrity of DNA
Timothy G. Standish, Ph. D.

3. DNA 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 cells DNA and foreign DNA
Distinguishing between old and new DNA strands
Controlling Gene Expression

4. Methylation N O NH2 CH3 5-Methyl cytidine N O NH2 Cytidine
5-Methylcytosine is the most commonly methylated nulceotide in E. coli. N O
NH2
CH3
5-Methyl
cytidine
Methylation N O
NH2
Cytidine 4 5 3 6 2 1
4-Methylcytosine is less common, but is also known.

5. Methylation N N6-Methyl adenine NH H3C NH2 Adenine N N N N
6-Methyladenine is another common methylated nulceotide.
Methylation N
N6-Methyl
adenine NH
H3C
NH2
Adenine N 6 N 7 5 1 8 4 2 9 N 3 N

6. E. coli 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

7. Destroying 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 cells DNA
Working with methylases, REs restrict bacteriophages to only one host bacterial strain.

8. Bacteriophage Attack Destruction of the bacteria’s DNA Infection
Replication of the viral genome
Production of viral parts
Packaging
Lysis

9. Repelling Bacteriophage Attack
Methylation sites
Methylase M

10. Repelling Bacteriophage Attack
Methylation sites
Unmethylated
methylation
sites R
Munch! Munch! Munch . . .

11. Repelling Bacteriophage Attack
Methylation sites
Take that you wicked virus!

12. Repelling Bacteriophage Attack
Methylaes and restriction endonucleases must recognize the same sequences if they are to function as an effective system
Take that you wicked virus!

13. Restriction Endonucleases
There are a number of different sub classes of restriction endonucleases
Type I - Recognize specific sequences and cut DNA a nonspecific site > than 1,000 bp away
Type II - Recognize palindromic sequences and cut within the palindrome
Type III - Recognize specific 5-7 bp sequences and cut bp down stream of the site.
Type II restriction endonucleases are the most useful class as they recognize specific palindomic sequences in DNA and cut the sugar phosphate backbone within the palindrome

14. 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.

15. DNA 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'

16. Uses of Type II Restriction Endonucleases
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.

17. R. E.s and DNA Ligase Can be used to make recombinant DNA
EcoRI
GAATTC
CTTAAG G
CTTAA
AATTC
1 Digestion
2 Annealing of sticky ends
3 Ligation
Ligase G
CTTAA
AATTC G G
CTTAA
AATTC
4 Recombinant DNA

18. Question
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

19. Mutation 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

20. Mutations Mutation = A random change in the genetic material of a cell
Two major types of mutations:
Macro mutations:
Chromosome number mutations
Addition or deletion of large chunks of DNA
Movement of large chunks of DNA
Point mutations:
Changes in only one or two bases in a gene
Not all mutations result in phenotypic change

21. Micro 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)

22. Frame Shift Mutations 3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’
3’AGTTCAG-TAC-TGA-AAC-CAT-CAA-CTG-ATCATC5’
5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Cys
Gly
Ser
5’AGUC-AUG-ACU-UUG-GUA-GUU-GAC-UAG-AAA3’
Met
Thr
Val
Leu
Frame shift mutations tend to have a dramatic effect on proteins as all codons down stream 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.

23. Substitution Mutations
Transition
3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Cys
Gly
Ser
Pyrimidine to
Pyrimidine
3’AGTTCAG-TAC-TGA-ATA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UAU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Gly
Ser
Tyr
Transversion
3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Cys
Gly
Ser
Purine to
Pyrimidine
3’AGTTCAG-TAC-TGA-AAA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UUU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Gly
Ser
Phe

24. Transitions 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

25. The Sickle Cell Anemia Mutation
Normal b-globin DNA T C A
Mutant b-globin DNA G U
mRNA A G
mRNA
Glu
Normal b-globin
Val
Mutant b-globin
H2N OH C O
H2C H
CH2
Acid
H2N OH C O
H3C H CH
CH3
Neutral
Non-polar
©1998 Timothy G. Standish

26. Sickle Cell Anemia: A Pleiotropic Trait
Mutation of base 2 in b globin codon 6 from A to T
causing a change in meaning from Glutamate to Valine
Mutant b globin is produced
Red blood cells sickle
Accumulation of sickled
Cells in the spleen
Clogging of small
Blood vessels
Breakdown of
Red blood cells
Heart failure
Pain and
Fever
Brain
damage
Damage to other organs
Spleen damage
Anemia
Weakness
Tower skull
Impaired
mental function
Infections
Especially
Pneumonia
Paralysis
Kidney
failure
Rheumatism

27. Repair 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

28. Direct 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 (ie thymine) together to form pyrimidine dimers
Photoreactivation of pyrimidine dimers is achieved by the detection of the primers by a light dependant 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

29. Thymine Dimers H P O HO
CH2 OH
NH2 N
CH3 HN UV
Light
Thymine

30. Thymine Dimers Photolyase Light Thymine CH3 HN O NH2 HO P N CH2 OH OH

31. Thymine Dimers
Photolyase H P O HO
CH2 OH
NH2 N
CH3 HN
Thymine

32. Thymine Dimers H P O HO
CH2 OH
NH2 N
CH3 HN
Thymine

33. Mutation When Mistakes Are Made5’
DNA
Pol. 5’ 3’
Mismatch 5’
DNA
Pol. 5’ 3’
3’ to 5’ Exonuclease activity
DNA
Pol. 5’ 3’

34. Mutation Excision Repair3’ 5’
Endo-
Nuclease
Thimine
Dimer 5’ 3’
Nicks
DNA
Pol. 5’ 3’
Ligase
DNA
Pol.
Ligase

35. The
End

36. Macromutations 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

37. Macromutation - Deletion
Chromosome
Centromere
A B C D E F G H
Genes
A B C D G H
E F

38. Macromutation - Duplication
Chromosome
Centromere
A B C D E F G H
Genes
A B C D E F E F G H
E F
Duplication

39. Macromutation - Inversion
Chromosome
Centromere
Genes
A B C D E F G H
Inversion
A B C D F E G H

40. Macromutation - Translocation
Chromosome
Centromere
Genes
A B C D E F G H
A B E F C D G H