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©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,

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Presentation on theme: "©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,"— Presentation transcript:

1 ©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, till all be fulfilled.

2 ©2001 Timothy G. Standish Repair: Maintaining the integrity of DNA Timothy G. Standish, Ph. D.

3 ©2001 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

4 ©2001 Timothy G. Standish Methylation 5-Methylcytosine is the most commonly methylated nulceotide in E. coli. 6 1 2 3 4 N O NH 2 O N N Cytidine 5 N O NH 2 N O CH 3 N 5-Methyl cytidine Methylation NH 2 4-Methylcytosine is less common, but is also known.

5 ©2001 Timothy G. Standish N N Adenine N N NH 2 Methylation 6-Methyladenine is another common methylated nulceotide. 1 3 4 7 6 2 8 9 5 Methylation N N N 6 -Methyl adenine N N NH H3CH3C

6 ©2001 Timothy G. Standish E. coli Methylation Systems Three methylation systems are known in E. coli: 1dcm system - Methylates cytosine - Function is unknown 2dam 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 3hsd 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 ©2001 Timothy G. Standish 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 cell’s DNA Working with methylases, REs restrict bacteriophages to only one host bacterial strain.

8 ©2001 Timothy G. Standish Lysis Bacteriophage Attack Destruction of the bacteria’s DNA Replication of the viral genome Production of viral parts Packaging Infection

9 ©2001 Timothy G. Standish Repelling Bacteriophage Attack Methylation sites M Methylase

10 ©2001 Timothy G. Standish Methylation sites Repelling Bacteriophage Attack Unmethylated methylation sites R Munch! Munch! Munch...

11 ©2001 Timothy G. Standish Repelling Bacteriophage Attack Methylation sites Take that you wicked virus!

12 ©2001 Timothy G. Standish Repelling Bacteriophage Attack Take that you wicked virus! Methylase and restriction endonucleases must recognize the same sequences if they are to function as an effective system

13 ©2001 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

14 ©2001 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.

15 ©2001 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'

16 ©2001 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.

17 ©2001 Timothy G. Standish G CTTAA AATTC G 1 Digestion 2 Annealing of sticky ends 3 Ligation Ligase G CTTAA AATTC G EcoRI R. E.s and DNA Ligase Can be used to make recombinant DNA GAATTC CTTAAG GAATTC CTTAAG G CTTAA AATTC G 4 Recombinant DNA

18 ©2001 Timothy G. StandishQuestion 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 ©2001 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

20 ©2001 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

21 ©2001 Timothy G. Standish Micro or Point Mutations Two major types of Micromutations are recognized: 1Frame Shift - Loss or addition of one or two nucleotides 2Substitutions - 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 ©2001 Timothy G. Standish Frame Shift Mutations 5’ AGUC-AUG-ACU-UUG-GUA-GUU-GAC-UAG-AAA 3’ 3’ AGTTCAG-TAC-TGA-AAC-CAT-CAA-CTG-ATCATC 5’ 3’ AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC 5’ 5’ AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA 3’ MetThrCys Gly Ser MetThrVal Leu 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.

23 ©2001 Timothy G. Standish Purine to Pyrimidine Transversion Pyrimidine to Pyrimidine Transition Substitution Mutations 3’ AGTTCAG-TAC-TGA-ATA-CCA-TCA-ACT-GATCATC 5’ 3’ AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC 5’ 5’ AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA 3’ MetThrCys Gly Ser 3’ AGTTCAG-TAC-TGA-AAA-CCA-TCA-ACT-GATCATC 5’ 3’ AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC 5’ 5’ AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA 3’ MetThrCys Gly Ser 5’ AGUC-AUG-ACU-UAU-GGU-AGU-UGA-CUAGAAA 3’ MetThr Gly Ser Tyr 5’ AGUC-AUG-ACU-UUU-GGU-AGU-UGA-CUAGAAA 3’ MetThr Gly Ser Phe

24 ©2001 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

25 Val Mutant  -globin H2NH2N OH C O H2CH2C H C CH 2 C O Acid Glu Normal  -globin TCT Normal  -globin DNA H2NH2N OH C O H3CH3C H C CH CH 3 Neutral Non-polar AGA mRNA TCA Mutant  -globin DNA AGU mRNA The Sickle Cell Anemia Mutation ©1998 Timothy G. Standish

26 ©2001 Timothy G. Standish Weakness Tower skull Impaired mental function Infections especially pneumonia ParalysisKidney failure Rheumatism Sickle Cell Anemia: A Pleiotropic Trait Mutation 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 and Fever Brain damage Damage to other organs Spleen damage Anemia Accumulation of sickled cells in the spleen Clogging of small blood vessels Breakdown of red blood cells

27 ©2001 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

28 ©2001 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 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

29 Thymine Dimers Thymine H P O HO O O CH 2 OH H P O HO O O CH 2 O O H H P OH O O CH 2 O O H HOH P O O O CH 2 NH 2 N N N CH 3 O O HN N N NH 2 N N N CH 3 O O HN N N

30 Thymine Dimers Thymine OH Photolyase

31 Thymine Dimers Thymine H P O HO O O CH 2 OH H P O HO O O CH 2 O O H H P OH O O CH 2 O O H HOH P O O O CH 2 NH 2 N N N CH 3 O O HN N N NH 2 N N N CH 3 O O HN N N Photolyase

32 Thymine Dimers Thymine H P O HO O O CH 2 OH H P O HO O O CH 2 O O H H P OH O O CH 2 O O H HOH P O O O CH 2 NH 2 N N N CH 3 O O HN N N NH 2 N N N CH 3 O O HN N N

33 ©2001 Timothy G. Standish Mutation When Mistakes Are Made 5’3’ 5’ DNA Pol. 5’ 3’ 5’3’ 5’ DNA Pol. DNA Pol. Mismatch 3’ to 5’ Exonuclease activity

34 ©2001 Timothy G. Standish Thimine Dimer 5’3’ 5’ Mutation Excision Repair 3’ 5’3’ 5’ 3’ 5’ DNA Pol. DNA Pol. Ligase Endo- Nuclease Ligase Nicks

35 ©2001 Timothy G. Standish

36 Macromutations Four major types of Macromutations are recognized: 1Deletions - Loss of chromosome sections 2Duplications - Duplication of chromosome sections 3Inversions - Flipping of parts of chromosomes 4Translocations - Movement of one part of a chromosome to another part

37 ©2001 Timothy G. Standish Macromutation - Deletion Chromosome Centromere A B C D E F G H Genes E F A B C D G H

38 ©2001 Timothy G. Standish Macromutation - Duplication A B C D E F E F G H Chromosome Centromere A B C D E F G H Genes E F Duplication

39 ©2001 Timothy G. Standish Macromutation - Inversion Chromosome Centromere A B C D F E G H Genes A B C D E F G H Inversion

40 ©2001 Timothy G. Standish Macromutation - Translocation A B E F C D G H Chromosome Centromere Genes A B C D E F G H

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