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.

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

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

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.

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

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

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.

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

Repelling Bacteriophage Attack Methylation sites Methylase M

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

Repelling Bacteriophage Attack Methylation sites Take that you wicked virus!

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!

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 24-27 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

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 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 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.

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

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

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

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

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)

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.

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

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

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

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

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

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

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

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

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

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

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

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

The End

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

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

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

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

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