biochemistry for pharmacy students nucleic acid structure & function
DESCRIPTION
Biochemistry for Pharmacy Students NUCLEIC ACID STRUCTURE & FUNCTION. Pál Bauer Dept. of Medical Biochemistry Rm. 4 515, EOK Email: [email protected]. OBJECTIVES. To learn the structures and properties of nucleotides, the building blocks of nucleic acids - PowerPoint PPT PresentationTRANSCRIPT
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Biochemistry for Pharmacy Students
NUCLEIC ACID STRUCTURE & FUNCTION
Pál Bauer
Dept. of Medical Biochemistry
Rm. 4515, EOK
Email: [email protected]
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OBJECTIVES
• To learn the structures and properties of nucleotides, the building blocks of nucleic acids
• To describe how the structure of DNA relates to functions of the genetic machinery.
• To explain how DNA is synthesized.• To describe how mutations in DNA can lead
to genetic diseases.• To explain how recombinant DNA technology
can be applied to the diagnosis and therapy of human genetic diseases (discussion sessions).
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HISTORICAL PERSPECTIVE
Nucleic acid structure & metabolism is directly relevant to cancer, gout, many genetically inherited diseases, AIDS and other infectious diseases.
The beginnings of nucleic acid biochemistry & genetics occurred in the 1860s:
- Frederic Miescher – Swiss biochemist who discovered nucleic acids.
- Gregor Mendel – Austrian monk who founded the science of genetics.
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Frederick Meischer studied salmon
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GregorMendelstudiedgardenpeas
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• Sugars and phosphates– Ribose in RNA– Deoxyribose in DNA
• Partial hydrolysis products– Nucleotides (contains
phosphates)
– Nucleosides (no phosphates)
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Names of nucleosides
Base Ribonucleoside Deoxyribonucleoside
Adenine Adenosine Deoxyadenosine
Guanine Guanosine Deoxyguanosine
Cytosine Cytidine Deoxycytidine
Uracil Uridine Deoxyuridine
Thymine Thymine riboside Thymidine
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Nucleosides: numbering system
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• The intra-nucleotide linkage
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Base composition of double stranded DNA
• [pyrimidines] = [purines]• Content: A = T, G = C• DNA base compositions are the same for
different tissues of the same organism.
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Watson-Crick base pairing
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Watson
Crick
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Animal Cell(eukaryote)
Plant Cell(eukaryote)
Bacterial Cell(prokaryote)
A Reminder of the Differences betweenEukaryotes and Prokaryotes
Fig. 2-7
E. coli
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Model of the Nuclear Envelope
Artwork by Don Guzy
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• The Watson-Crick Structure
• Right-handed helices• Anti-parallel• Base-pairing• Open structure accessible
to water• Stacking forces between
planar paired bases give a rigid structure
5’ 3’
3’5’
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Denaturation of DNABy pH, heat, solvents, urea, amides• Helix-coil transition• Hyperchromic effect• Melting temperature
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Denaturation ofdouble-stranded
DNA
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Indirect evidencea. High DNA content of chromosomesb. 260 nm is a very mutagenic wavelength;
bases maximally absorb light energy at 260 nm
c. Constancy of [DNA] / cell (germ cells) and 2x [DNA] / cell (somatic cells)
DNA IS THE GENETIC MATERIAL IN CELLS
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a. Transformation of bacteria with DNA
Requires both DNA functions:
replication and expression
b. Transfection with viral nucleic acids
c. In vitro expression of DNA (from bacteriophage T4)
d. Synthesis of active DNA in vitro
Direct Evidence
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Pneumococci
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DNA CONTENT
OF SOME CELLS AND VIRUSES
Haploid size of genome, Source of DNA base pairs____ Viruses
SV40 5 x 103 (5 kb)Papilloma (wart) 8 x 103
Adenoviruses 2.1 x 104
Herpesviruses 1.56 x 105
Poxviruses 2.4 x 105 Cells
Escherichia coli 4.5 x 106 (4,500 kb)Yeast 1.3 x 107
Drosophila 1.6 x 108
Human 3.2 x 109 (3.2x106 kb)Frogs 6 x 109 (6 x 106 kb)Onion Plant 18 x 109
Fern Plant 160 x 109
Animal mitochondria 1.5 x 104 (15 kb)Plant chloroplast 1 x 105
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The total length of the circular E. coli chromosome is 1.7 mm,whereas the length of an E. coli cell is 2 μm.
partially lyzedE. coli cell
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22 autosomal chromosomes (chromosomes 1-22)
plus X and Y chromosomes
3.2 x 109 total bp
Human haploid genome
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• Histones and chromosome structure
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a. Nucleosomes
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The double-stranded DNAis wrappedaround theoutside of eachnucleosome twice.
The nucleosomesare regularly spaced along theDNA.
electron micrograph of chromatin
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electron micrograph of supercoiled nucleosomesin chromatin.
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A typical phone cord is coiledlike a DNA helix, and thecoiled cord can itself coil in in a supercoil. If twistedtight enough, the supercoilswill themselves form an evenhigher order of supercoiling.
Double-stranded DNA helicesalso form supercoils of
supercoils.
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Two drawings depicting the differentlevels of DNA supercoiling that provideDNA compaction in a eukaryoticchromosome. The levels take the formof coils upon coils.
histones
nucleosomes
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5’ 3’
DNA replication
5’ 3’
3’3’5’5’ 3’5’
5’3’
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5’
5’3’
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All DNA polymerases catalyze elongation ofthe primer strand in a 5’ to 3’ direction, copying the template strand in a 3’ to 5’direction. This means that the “leading strand” can be synthesized continuously, but the “lagging strand” must be synthesized discontinuously.
A problem is that DNA polymerases must have a primer strand with a 3’ OH from whichto begin DNA synthesis. So, wheredo these primers come from whena DNA polymerase synthesizes newOkazaki fragments in the laggingstrand?
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5’ 3’
5’3’
5’5’ 3’3’
DNA
Replication does not usually begin at the end of a DNA molecule; itbegins in the middle of the DNA.
(DNA pol I, II and III)
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12.4 Schematic model of the proofreading function of DNA
polymerase
Figure 12-21
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An example of error correction by the 3’ to 5’
exonuclease (proofreading)activity of DNA polymerase I.
A mismatched base pair (a C-A mismatch) impedes the
movement of DNA polymerase Ito the next site. Sliding back-ward, the enzyme removes itsmistake with the 3’ to 5’ exo-
nuclease activity & thenresumes its 5’ to 3’
polymerase activity.
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c. DNA Polymerase II
• Mutants in the E. coli gene for this DNA polymerase are not lethal. Also is a repair enzyme. • Requires duplex DNA template and primer. • Utilizes a template strand and elongates a primer
strand, similar to DNA polymerase I. d. DNA Polymerase III (pol C gene)
Mutants are ts (temperature sensitive), i.e., conditionally lethal.
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The 3-D structure of E. coli DNA polymerase I.
The active site for the polymerase activity and the 3’ to 5’ exonucelase activity is deep in the crevice at the far end of the bound DNA. The template strandis dark blue.
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a. Illustration of the 5’ to 3’exonuclease activity of E. coliDNA polymerase I (sometimescalled a “nick translation”activity).
An RNA or DNA strand paired with a DNA templatestrand is simultaneously degraded by the 5’ to 3’ exonuclease activity & is replaced by the polymeraseactivity of the same enzyme.
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b. The origin of replication – the ori C locus Accessory proteins
dna B gene productProbably this protein is membrane-
associated and recognizes the initiation sequence on DNA.
c. RNA primersDNA polymerases require a preformed
primer; RNA polymerases do not.
1. Accessory protein binds (dna B protein) 2. "Primase," an RNA polymerase (dna G)
It does not require a primer; forms a short piece of RNA complementary to the DNA
template strand.
3. DNA binding proteins
d. Details of the events:
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cellmembrane
cellmembrane
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5’
5’
5’3’
5’3’
5’3’
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The 5’ end of RNA primer is usually 5’ pppA... or 5’ pppG….
DNA polymerase III dissociates the primase that synthesizes the RNA primer.
The 5' → 3' nuclease of DNA polymerase l, or another enzyme called RNase H, removes the RNA primer. RNase H hydrolyzes RNA in DNA/RNA base-paired regions.
DNA polymerase I (and maybe DNA pol II?) fill the gaps left by the removal of the RNA primers.
The complete but "nicked" (broken) daughter strands that result after the gaps are filled in are then joined by DNA ligase.
Additional information about the overall process of DNA replication
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• DNA ligase (animal enzyme)
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• The unwinding problem: 5000 turns/min in E. coli (helicases and topoisomerases)
Topoisomerase I: relaxes supercoils
a. Enzyme recognizes right-handed superhelices
introduced during DNA replication.
b. Small DNA segment unwinds, as strand breaks, the superhelix relaxes.
c. Enzyme, still attached to broken strand, reseals the break.
d. The topoisomerase is both a nuclease and a ligase. (The nucleolytic activity can be viewed as the reverse of ligase action.)
Helicases: unwind the two strands of DNA & cause supercoiling ahead of the fork.
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SUMMARY OF DNA REPLICATION IN BACTERIA
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Map of the E. coli genome showing some of the genes that encodeproteins involved in DNA replication, repair and recombination.
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DNA REPLICATION in EUKARYOTIC CELLS is SOMEWHATMORE COMPLICATED and INVOLVES ADDITIONAL DNA
POLYMERASES & MULTIPLE REPLICATION ORIGINS
Multiple replication origins occur in the large eukaryotic chromosomes.
Greek
Name
Gene
Name
Proposed Main Function
alpha POLA DNA Replication
beta POLB Base Excision Repair
gamma POLG Mitochondrial Replication
delta POLD1 DNA Replication
epsilon POLE DNA Replication
zeta POLZ Bypass Synthesis
eta POLH Bypass Synthesis
theta POLQ DNA Repair
iota POLI Bypass Synthesis
kappa POLK Bypass Synthesis
lambda POLL Base Excision Repair
mu POLM Non-Homologous End Joining
sigma POLS Sister Chromatid Cohesion
• The number of known eukaryotic DNA polymerases has doubled in the last several years as researchers discover additional enzymes with DNA polymerizing activity.
• Most of the newly discovered DNA polymerases are involved in repair of DNA, rather than DNA replication.
• The DNA polymerases alpha, delta and epsilon are the most clearly involved in DNA replication.
• DNA polymerases delta and epsilon have proof-reading 3’->5’ exonuclease activity; the other DNA polymerases do not.
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Types and rates of mutation
Type Mechanism Frequency________ Genome chromosome 10-2 per cell division mutation missegregation
(e.g., aneuploidy)
Chromosome chromosome 6 X 10-4 per cell division mutation rearrangement
(e.g., translocation)
Gene base pair mutation 10-10 per base pair per mutation (e.g., point mutation, cell division or
or small deletion or 10-5 - 10-6 per locus per insertion generation
Mutation
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Mutation rates* of selected genes
Gene New mutations per 106 gametes
Achondroplasia 6 to 40Aniridia 2.5 to 5Duchenne muscular dystrophy 43 to 105Hemophilia A 32 to 57Hemophilia B 2 to 3Neurofibromatosis -1 44 to 100Polycystic kidney disease 60 to 120Retinoblastoma 5 to 12
*mutation rates (mutations / locus / generation) can varyfrom 10-4 to 10-7 depending on gene size and whetherthere are “hot spots” for mutation (the frequency at mostloci is 10-5 to 10-6).
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Many polymorphisms exist in the genome
• the number of existing polymorphisms is ~1 per 500 bp• there are ~5.8 million differences per haploid genome• polymorphisms were caused by mutations over time• polymorphisms called single nucleotide polymorphisms
(or SNPs) are being catalogued by the HumanGenome Project as an ongoing project
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Types of base pair mutations
CATTCACCTGTACCAGTAAGTGGACATGGT
CATGCACCTGTACCAGTACGTGGACATGGT
CATCCACCTGTACCAGTAGGTGGACATGGT
transition (T-C to A-G) transversion (T-A to G-C)
CATCACCTGTACCAGTAGTGGACATGGT
deletionCATGTCACCTGTACCAGTACAGTGGACATGGT
insertion
base pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purine
normal sequence
deletions and insertions can involve one or more base pairs
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Spontaneous mutations can be caused by tautomers
Tautomeric forms of the DNA bases
Adenine
Cytosine
AMINO IMINO
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Guanine
Thymine
KETO ENOL
Tautomeric forms of the DNA bases
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Mutation caused by tautomer of cytosine
Cytosine
Cytosine
Guanine
Adenine
• cytosine mispairs with adenine resulting in a transition mutation
Normal tautomeric form
Rare imino tautomeric form
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Mutation is perpetuated by replication
• replication of C-G should give daughter strands each with C-G
• tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strands
• which could result in a C-G to T-A transition mutation in the next round of replication, or if improperly repaired
C G C G
C G C A
AC T A
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Chemical mutagens
Deamination by nitrous acid
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N
NH
NH
N
NH2
O
N
NH
NH
NH
NH2
O
O
Attack by oxygen free radicalsleading to oxidative damage
guanine
8-oxyguanine (8-oxyG)
• many different oxidative modifications occur• by smoking, etc.• 8-oxyG causes G to T transversions
• the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation• mutation of the MTH1 gene causes increased tumor formation in mice
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Ames test for mutagen detection
• named for Bruce Ames• reversion of histidine mutations by test compounds• His- Salmonella typhimurium cannot grow without histidine• if test compound is mutagenic, reversion to His+ may occur• reversion is correlated with carcinogenicity
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Thymine dimer formation by UV light
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Summary of DNA lesions
Missing base Acid and heat depurination (~104 purinesper day per cell in humans)
Altered base Ionizing radiation; alkylating agents
Incorrect base Spontaneous deaminationscytosine to uraciladenine to hypoxanthine
Deletion-insertion Intercalating reagents (acridines)
Dimer formation UV irradiation
Strand breaks Ionizing radiation; chemicals (bleomycin)
Interstrand cross-links Psoralen derivatives; mitomycin C
Tautomer formation Spontaneous and transient
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DNA repair activity
Life
sp
an
1
10
100 human
elephant
cow
hamsterratmouseshrew
Correlation between DNA repairactivity in fibroblast cells fromvarious mammalian species andthe life span of the organism
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Defects in DNA repair or replicationAll are associated with a high frequency of chromosome
and gene (base pair) mutations; most are also associated with a predisposition to cancer, particularly leukemias
• Xeroderma pigmentosum• caused by mutations in genes involved in nucleotide excision repair• associated with a >1000-fold increase of sunlight-induced skin cancer and with other types of cancer such as melanoma
• Ataxia telangiectasia• caused by gene that detects DNA damage• increased risk of X-ray• associated with increased breast cancer in carriers
• Fanconi anemia• caused by a gene involved in DNA repair• increased risk of X-ray and sensitivity to sunlight
• Bloom syndrome• caused by mutations in a a DNA helicase gene• increased risk of X-ray• sensitivity to sunlight
• Cockayne syndrome• caused by a defect in transcription-linked DNA repair• sensitivity to sunlight
• Werner’s syndrome• caused by mutations in a DNA helicase gene• premature aging
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Transition vs. transversion• TransitionExchange of a purine with a purine or pyrimidine with a
pyrimindine base. More common than transversion. Often the result of tautomeric shifts.
– GC AT transition • causal agents: e.g. base analog 5’bromouracil
– AT GC transition • causal agents: e.g. base analog 2-aminopurine
• TransversionExchange of a purine with a pyrimidine base and vice versa– GC TA or GC CG transversion– AT CG or AT TA transversion
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Methyl-directed mismatch repairCH3 CH3
CH3 CH3
CH3 CH3MutS
MutL
CH3 CH3
MutL
MutSMutHMutH
MutS, MutL, ATPADP+Pi
MutH, ATPADP+Pi
1. Mismatch within 1 kb ofmethylated GATC
2. MutS and MutH bind to mismatched spots along the DNA (except C-C)
3. DNA on both sides of theMitsmatch runs throughMutS:MutL complex
4. MutH binds to MutL andto GATC
5. Endonuclease of MutH cleaves unmethylated DNA at hemimethylated GATC
5’3’
5’3’
5’
5’
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Mismatch repair in E. coliScenario 1Mismatch is at the 5’ end of cleavage site
1. Unmethylated DNA is unwound via DNA helicase II
2. The 3’-5’ exonuclease activity of exonuclease I or exo X degrades DNA through the mismatch
3. DNA polymerase III synthesizes the new DNA strand
4. DNA ligase closes the remaining nick.
CH3 CH3
ATPADP+Pi
MutS-MutLDNA helicase IIexonuclease I
orexonuclease X
CH3 CH3
CH3 CH3
5’3’
5’3’
5’3’
3’5’
3’5’
3’5’
DNA polymerase IIISSBs
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Mismatch repair in E. coliCH3 CH3
ATPADP+Pi
MutS-MutLDNA helicase IIexonuclease VII
orRecJ nuclease
CH3 CH3
CH3 CH3
Scenario 2Mismatch is at the 3’ end of cleavage site
1. Unmethylated DNA is unwound via DNA helicase II
2. The 5’-3’ exonuclease activity of exonuclease VII or RecJ nuclease degrades DNA through the mismatch
3. DNA polymerase III synthesizes the new DNA strand.
4. DNA ligase closes the remaining nick.
5’3’
5’3’
5’3’
3’5’
3’5’
3’5’
DNA polymerase IIISSBs
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Base excision repair• not restricted to a short time post replication• similar in most organisms (bacteria – mammals)• recognizes abnormal bases in the DNA• usually less expensive than mismatch repair• requires four enzymes
1. DNA glycosylases
2. AP-endonucleases
3. DNA polymerase I
4. DNA ligase
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DNA glycosylases• Relatively small enzymes (20 – 30 KDa)
• Recognize abnormal bases – deaminated bases– alkylated bases
• Remove base via cleavage at the glycosidic bond between the deoxyribose and the base
• Cleavage creates apurinic and apyrimidinic (AP sites)
UOP
GOP
AOP
O
GOP
AOP
P
Before After
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DNA glycosylasesEnzyme Units / mg protein (x 10 - 3)
not adapted adaptedUracil DNA glycosylase 3,800 3,500
Hypoxanthin DNA glycosylase 2.1 23- methyladenine I DNA glycosylase 3.6 3.6
3- methyladenine II DNA glycosylase (*) 0.22 4.1Formamidopyrimidine DNA glycosylase 4.2 3.9
Legend:Adapted = incubation with 1 g/ml NNG for 1 hourNot adapted = untreated control
Source: Karran et al. (1983), Nature 296, 770 - 773
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AP-endonucleases• recognize AP-sites• cleave phosphodiester
bonds near the AP site and generate a 5’ –phosphate and 3’-hydroxyl
• In E. coli this enzyme also has 3’-5’ exonuclease activity
• The 3’-OH functions as a primer
P
A
P
G
P
G
P
C
P
T
P P
C
P
P
A
P
G
P
C
P
T
P
C
P
GC
P
A
P
G
P
G
P
C
P
T
P
P
A
P
G
P
C
P
C
P
GC
AP endonuclease
5’
3’
5’3’
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Base excision repair (BER)
P
A
P
G
P
G
P
C
P
T
P P
C
P
G
P
A
P
G
P
G
P
C
P
T
P
U
P
C
P
G
P
A
P
G
P
G
P
C
P
T
P
G
P
A
P
G
P
G
P
C
P
T
P
C
P
C
P
G
Uracil DNA glycosylase
AP endonuclease
DNA polymerase I
DNA ligase
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Defects in BER• In humans BER involves:
– DNA polymerase beta– The Xrcc1 geneproduct– Ape1 (first step in removing the damaged base)
• BER deficiencies have been implicated with: – cancer– neurodegenerative diseases– aging
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Nucleotide excision repair• Recognizes large distortions in the DNA structure• Repairs UV-damaged DNA• Multisubunit enzyme • Cleaves two phosphodiester bonds upstream and
downstream of the lesion• Generally generates fragments of 12 to 13 nts• Requires four different enzymes
1. Exinuclease
2. DNA helicase
3. DNA polymerase
4. DNA ligase
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Nucleotide excision repair in E. coliEnzyme Protein Function
UvrA (MW= 104,000) scans DNA, binds to UvrB
UvrB (MW = 78,000)scanner; binds DNA cleaves phosphate bond at 3' end, 5
positions downstream of lesion
UvrC (MW = 68,000) binds UvrB & DNA cleaves phosphate bond at 5' end, 8 positions upstream of lesion
DNA helicase UvrD removes DNA fragmentDNA
polymeraseDNA polymerase I
(= PolA )fills emerging gap
DNA ligase Lig seal nick
Exinuclease
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Nucleotide excision repair in E. coli
exinuclease
UvrD DNA helicase
DNA pol. I DNA ligase
ATP
PP
POH
PP
UvrA UvrBUvrAMechanism
• The (UvrA)2:UvrB complex scans DNA
• UvrA dimer dissociates from pryimidine dimer. UvrB binds DNA and cuts at 3’ end.
• UvrC associates with UvrB and cuts DNA at 5’ end of the pyrimidine dimer
• UvrD DNA helicase removes the DNA fragment
• DNA polymerase I fills the gap• DNA ligase seals the remaining
nick.
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Xeroderma pigmentosum• Cause:
– Defect in human nucleotide excision repair (NER)– 16 polypeptides involved in NER– NER is the only pathway to remove pyrimidine dimers
in humans
• Symptoms:– Very light sensitive– High risk of sun-light induced skin cancer– Neurological abnormalities (high rate of oxidative
metabolism in neurons)
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Direct repair• Observation:
– UV-damaged bacteria that were subsequently incubated in daylight recovered better than those kept in the dark
• Photoreactivation– requires DNA photolyases– requires visible light at 300 – 500 nm– aka “light repair” – Contrast: dark repair (BER, NER, mismatch repair)
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DNA photolyases• Structure
– MW = ~ 54,000 (in E. coli)– Generally contain 2 chromophores
– Chromophore No. 1: always FADH -
– Chromophore No. 2: folate (in E. coli and yeast) • N5,N10-methenyltetrahydrofolylpolyglutamate
• Function– bind to pyrimidine dimers– resolve pyrimidine dimers into original bases
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Mechanism outline1. Absorption of a photon by MTHFpolyGlu
2. Transfer of excitation energy to FADH –
3. Excited FADH – transfers elecctron to the pyrimidine dimer (unstable dimer radical)
4. Shift of electrons breaks cyclobutane ring
5. Electron is transferred back to the flavin radical to regenerate FADH –
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O6-methylguanine• Mutagenic behavior
– pairs with thymine rather than cytosine
• Specific Repair Mechanism – differs from base excision repair– requires O6-methylguanine methyltransferase– one time reaction (“suicide enzyme”)– expensive repair mechanism– methylated methyltransferase is a transcriptional
activator of its own gene
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O6-methylguanine DNA methyltransferase
N
NN
NH2N
O-CH3
R
N
NN
NH2N
O
R
active Enz.
Cys-SH
active Enz.
Cys-S-CH3
Source: adapted from Lehninger pg. 957
O6-methylguanine nucleotide guanine nucleotide
H H
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Transition vs. transversion• TransitionExchange of a purine with a purine or pyrimidine with a
pyrimindine base. More common than transversion. Often the result of tautomeric shifts.
– GC AT transition • causal agents: e.g. base analog 5’bromouracil
– AT GC transition • causal agents: e.g. base analog 2-aminopurine
• TransversionExchange of a purine with a pyrimidine base and vice versa– GC TA or GC CG transversion– AT CG or AT TA transversion
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O6-methylguanine• Mutagenic behavior
– pairs with thymine rather than cytosine
• Specific Repair Mechanism – differs from base excision repair– requires O6-methylguanine methyltransferase– one time reaction (“suicide enzyme”)– expensive repair mechanism– methylated methyltransferase is a transcriptional
activator of its own gene
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O6-methylguanine DNA methyltransferase
N
NN
NH2N
O-CH3
R
N
NN
NH2N
O
R
active Enz.
Cys-SH
active Enz.
Cys-S-CH3
Source: adapted from Lehninger pg. 957
O6-methylguanine nucleotide guanine nucleotide
H H
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Inaccurate DNA repairE. coli recA+ strain E. coli recA- strain
Numerous survivorsHigh mutation rates
Few survivorsLow mutation rates
intensive UV exposure
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Error-prone repair• Activated upon:
– severe DNA damage– disruption of DNA replication
• “SOS-response”– Inaccurate repair mechanism– Requires at least 14 proteins in E. coli
1. Din proteins (damage induced)
2. Rec poteins (recombination)
3. Umu proteins (UV-mutagenesis)
4. Uvr proteins (UV-resistance)
5. Others: SulA, HimA, Ssb, and PolB
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Genes involved in the SOS response
Gene Protein function encoded:pol B DNA polymerase II (polymerization subunit)uvrA & uvrB ABC exinucleaseumuC & umuD DNA polymerase Vsul A inhibits cell division via interaction with FtsZsul BrecA recA protease, recombination and repairdinB DNA polymerase IVssb single-stranded binding proteinuvrD DNA helicase II (DNA -unwinding protein)him A subunit of integration host factorrec N required for recombinational repairdinD ?dinF ?
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Damaged DNA during replication• UV-damaged DNA results in collapse of the replication fork during DNA
replication• 2 scenarios
– Unrepaired lesions Unrepaired DNA breaks
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12.4 DNA damage and repair and their role in carcinogenesis
• A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiation
• If uncorrected, such changes may interfere with the ability of the cell to function
• DNA damage can be repaired by several mechanisms • All carcinogens cause changes in the DNA sequence and
thus DNA damage and repair are important aspects in the development of cancer
• Prokaryotic and eukaryotic DNA-repair systems are analogous
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12.4 General types of DNA damage and causes
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12.4 Proofreading by DNA polymerase corrects copying errors
Figure 12-20
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12.4 Schematic model of the proofreading function of DNA
polymerase
Figure 12-21
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12.4 Chemical carcinogens react with DNA directly or after activation, and the
carcinogenic effect of a chemical correlates with its mutagenicity
Figure 12-22
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12.4 Base deamination leads to the formation of a spontaneous point
mutation
Figure 12-23
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12.4 Mismatch repair of single-base mispairs
Figure 12-24
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12.4 Chemically modified bases, such as thymine-thymine dimers, are corrected by
excision repair
Figure 12-25
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12.4 Excision repair of DNA by the E. coli UvrABC mechanism
Figure 12-26
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12.4 End-joining repair of nonhomologous DNA
Figure 12-28
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12.5 Recombination between homologous DNA sites
• Recombination provides a means by which a genome can change to generate new combinations of genes
• Homologous recombination allows for the exchange of blocks of genes between homologous chromosomes and thereby is a mechanism for generating genetic diversity
• Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sites is proportional to the distance between the sites
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12.5 The cross-strand Holliday structure is an intermediate in recombination (part
I)
Figure 12-29
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12.5 The cross-strand Holliday structure is an intermediate in recombination (part
II)
Figure 12-29