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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. ©2000 Timothy G. Standish Restriction and Repair: Maintaining the integrity of DNA Timothy G. Standish, Ph. D. ©2000 Timothy G. Standish 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 cell’s DNA and foreign DNA – Distinguishing between old and new DNA strands – Controlling Gene Expression ©2000 Timothy G. Standish Methylation 5-Methylcytosine is the most commonly methylated nulceotide in E. coli. 5 6 NH2 Cytidine 4 3N 1 N Methylation CH3 NH2 5-Methyl cytidine N 2 O N O 4-Methylcytosine is less common, but is also known. ©2000 Timothy G. Standish Methylation 6-Methyladenine is another common methylated nulceotide. H3C NH2 Adenine N7 5 6 1 N 8 9 N 4 3 N 2 Methylation N6-Methyl NH adenine N N N N ©2000 Timothy G. Standish 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 ©2000 Timothy G. Standish Destroying Foreign DNA Methylase enzymes methylate specific bases in specific sequences of DNA Only the cell’s own DNA is methylated at a given sequence Thus it is possible to differentiate between the cell’s 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 ©2000 Timothy G. Standish Bacteriophage Attack Infection Destruction of the bacteria’s DNA Production of viral parts Lysis Packaging Replication of the viral genome ©2000 Timothy G. Standish Repelling Bacteriophage Attack Methylation sites Methylase M ©2000 Timothy G. Standish Repelling Bacteriophage Attack Methylation sites Unmethylated methylation sites R Munch! Munch! Munch . . . ©2000 Timothy G. Standish Repelling Bacteriophage Attack Methylation sites Take that you wicked virus! ©2000 Timothy G. Standish Repelling Bacteriophage Attack Methylase and restriction endonucleases must recognize the same sequences if they are to function as an effective system Take that you wicked virus! ©2000 Timothy G. Standish Restriction Endonucleases There are a number of different subclasses 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 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 ©2000 Timothy G. Standish 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. ©2000 Timothy G. Standish 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' ©2000 Timothy G. Standish 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. ©2000 Timothy G. Standish R. E.s and DNA Ligase Can be used to make recombinant DNA EcoRI EcoRI GAATTC CTTAAG GAATTC CTTAAG G CTTAA 1 Digestion AATTC G 2 Annealing of sticky ends Ligase G AATTC CTTAA G 3 Ligation 4 Recombinant DNA G AATTC CTTAA G ©2000 Timothy G. Standish 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 ©2000 Timothy G. Standish 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 ©2000 Timothy G. Standish Mutations 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 ©2000 Timothy G. Standish 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) ©2000 Timothy G. Standish Frame Shift Mutations 3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’ 5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’ Met Thr Cys Gly Ser 3’AGTTCAG-TAC-TGA-AAC-CAT-CAA-CTG-ATCATC5’ 5’AGUC-AUG-ACU-UUG-GUA-GUU-GAC-UAG-AAA3’ Met Thr Leu Val Val Val 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. ©2000 Timothy G. Standish Substitution Mutations 3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’ 5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’ Transition Met Thr Cys Gly Ser 3’AGTTCAG-TAC-TGA-ATA-CCA-TCA-ACT-GATCATC5’ 5’AGUC-AUG-ACU-UAU-GGU-AGU-UGA-CUAGAAA3’ Met Thr Tyr Gly Ser Pyrimidine to Pyrimidine 3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’ 5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’ Transversion Met Thr Cys Gly Ser 3’AGTTCAG-TAC-TGA-AAA-CCA-TCA-ACT-GATCATC5’ 5’AGUC-AUG-ACU-UUU-GGU-AGU-UGA-CUAGAAA3’ Met Thr Phe Gly Ser Purine to Pyrimidine ©2000 Timothy G. Standish 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 ©2000 Timothy G. Standish The Sickle Cell Anemia Mutation Normal b-globin DNA C Mutant b-globin DNA T T C G A A G U A mRNA mRNA Normal b-globin Mutant b-globin Glu H2 N C C A T Val O OH H CH2 H2C C OH O Acid H2 N C C O OH H CH CH3 H3C Neutral Non-polar ©1998 Timothy G. Stan 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 Breakdown of red blood cells Anemia Clogging of small blood vessels Tower skull Weakness Heart failure Impaired mental function Accumulation of sickled cells in the spleen Red blood cells sickle Brain damage Paralysis Pain and Fever Damage to other organs Rheumatism Kidney failure Spleen damage Infections especially pneumonia ©2000 Timothy G. Standish 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 ©2000 Timothy G. Standish 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 (i.e., 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 ©2000 Timothy G. Standish Thymine Dimers OH HO P NH2 O N O CH2 OH Thymine N N O H N O CH2 O O N O CH2 N O OH H Thymine N N O CH2 H OH P O OH O P O HO NH2 OH P O H O H Thymine Dimers OH P NH2 O N O CH2 N N O H N Photolyase HO OH O Thymine CH2 O O N O CH2 N O N N OH H O Thymine CH2 H OH P O OH O P O HO NH2 OH P O H O H Thymine Dimers OH P NH2 O N O CH2 N N O H N Photolyase HO OH O Thymine CH2 O O N O CH2 N O N N OH H O Thymine CH2 H OH P O OH O P O HO NH2 OH P O H O H Thymine Dimers OH HO P NH2 O N O CH2 OH Thymine N N O H N O CH2 O O N O CH2 N O OH H Thymine N N O CH2 H OH P O OH O P O HO NH2 OH P O H O H Mutation When Mistakes Are Made 5’ DNA Pol. 5’ 5’ DNA Pol. 3’ to 5’ Exonuclease activity 5’ 3’ DNA Pol. 3’ 5’ 3’ 5’ ©2000 Timothy G. Standish Mutation Excision Repair 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ EndoNuclease 5’ Nicks DNA Ligase Pol. 3’ 5’ ©2000 Timothy G. Standish ©2000 Timothy G. Standish Macromutations 1 2 3 4 Four major types of Macromutations are recognized: Deletions - Loss of chromosome sections Duplications - Duplication of chromosome sections Inversions - Flipping of parts of chromosomes Translocations - Movement of one part of a chromosome to another part ©2000 Timothy G. Standish Macromutation - Deletion Chromosome Centromere Genes A B C D E F A B C D G H G H E F ©2000 Timothy G. Standish Macromutation - Duplication Chromosome Centromere Genes A B C D E F G H A B C D E F EE FF G H Duplication ©2000 Timothy G. Standish Macromutation - Inversion Chromosome Centromere Genes A B C D E F A B C D F E Inversion G H G H ©2000 Timothy G. Standish Macromutation - Translocation Chromosome Centromere A B C A B E Genes D F E C F G H D G H ©2000 Timothy G. Standish