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◦ DNA replication◦ DNA repair◦ DNA OrganizationKey topics:
 While functioning as a stable storage of genetic information,the structure of DNA is far from static:◦ A new copy of DNA...
 The Meselson-Stahl experimentwas about the origin of the twostrands in each of the daughtergenomes Cells were grown on ...
 The Meselson-Stahlexperiment showed that thenitrogen used for thesynthesis of new dsDNAbecomes equally dividedbetween th...
 Both strands are replicated simultaneously
 Parental DNA strand servesas a template Nucleotide triphosphatesserve as substrates in strandsynthesis 3’ HydroxylPrim...
 Polymerase I is most abundant but itsprimary function is in clean-up duringreplication, repair, and recombination Polym...
 Initiation◦ Requires initiator proteins (trans-acting factors) Elongation◦ Leading and Lagging strands (repeated primin...
 DNA Primase Synthesizes Short RNA PrimerMolecules on the Lagging Strand Helicases - Open Up the DNA Double Helixin Fron...
 Chemical reactions and some physical processes constantlydamage genomic DNA◦ At the molecular level, damage usually invo...
 Mismatches arise from occasional incorporationof incorrect nucleotides Abnormal bases arise from spontaneousdeamination...
The fundamental difference betweenprokaryotes and eukaryotes is thatprokaryotes have a single type ofchromosome, while mos...
•The complete set of all metaphasechromosomes in a cell is called itskaryotype•Karyotypes are species specific, and cellso...
•Human karyotypes show chromosomesarranged in order according to size andposition of the centromere•Karyotypes allow genet...
Example of the human karyotypeExample of the human karyotype11 22 33 44 5566 77 88 99 1010 1111 12121313 1414 1515 1616 17...
• Certain regions called bands onchromosomes stain more intensely thatother regions• Banding patterns are specific for eac...
Example of the human karyotypeExample of the human karyotype11 22 33 44 5566 77 88 99 1010 1111 12121313 1414 1515 1616 17...
• Q-banding produces bands whenchromosomes are stained with quinacrinedye, which binds preferentially to AT-richregions of...
Designations ofDesignations ofthe bands andthe bands andinterbands in theinterbands in thehuman karyotypehuman karyotypeG ...
• An organism’s total DNA content is called itsC-value: total amount of DNA in a haploid cell• The amount of genetic mater...
No direct relationshipNo direct relationshipbetween the C value andbetween the C value andthe structural orthe structural ...
• The large amount of DNA present ineukaryotic chromosomes is compacted byassociation with histones, formingstructures cal...
HISTONES•Small basic proteins•Constant amount in cells•25% lysine & arginine (Net + charge)•5 main types: H1, H2A, H2B, H3...
•Equal amount of histones & DNA•H2A, H2B, H3 & H4 are highly conservedamong distinct species•Histone proteins are among th...
NON-HISTONE PROTEINS•All DNA chromosomal proteins minushistones•Structural proteins or enzymes i.e. DNAreplication enzymes...
• Acidic proteins (negatively charged)• Equal amount of non-histones & DNA• Example of HMGs (High-Motility Group proteins)...
• Octamer of histones 2 (H2A, H2B, H3, H4)+ linker histone H1 + 180 bp of DNA• DNA compacts by winding 1 and ¾ turn ofthe ...
Nucleosome StructureNucleosome Structure
Nucleosomes connected together by linkerNucleosomes connected together by linkerDNA and H1 histone to produce the “beads-D...
Packaging of nucleosomes into the 30-nmPackaging of nucleosomes into the 30-nmchromatin fiberchromatin fiber
The many different ordersThe many different ordersof chromatin packing thatof chromatin packing thatgive rise to the highl...
• The functional state of thechromosome is related to the extent ofcoiling• The more condensed areas of thechromosome (het...
• Centromeres are the sites at whichchromosomes attach to the mitotic andmeiotic spindles• Consensus yeast centromeric reg...
The CentromereThe Centromere
• Telomeres are regions found at the end ofchromosomes• They are often associated with the nuclearenvelope and are common ...
Centromere and TelomeresCentromere and Telomeres
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
DNA metabolism and organization
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DNA metabolism and organization

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DNA metabolism and organization

  1. 1. ◦ DNA replication◦ DNA repair◦ DNA OrganizationKey topics:
  2. 2.  While functioning as a stable storage of genetic information,the structure of DNA is far from static:◦ A new copy of DNA is synthesized with high fidelity before each celldivision◦ Errors that arise during or after DNA synthesis are constantlychecked for, and repairs are made◦ Segments of DNA are rearranged either within a chromosome orbetween two DNA molecules giving offspring a novel DNA DNA metabolism consists of a set of enzyme catalyzed andtightly regulated processes that achieve these tasks
  3. 3.  The Meselson-Stahl experimentwas about the origin of the twostrands in each of the daughtergenomes Cells were grown on a mediumcontaining only 15N isotope untilall their DNA became fully 15Nlabeled Cells were then switched to 14Nmedium and allowed to divideonce CsCl density gradientcentrifugation was used todetermine the mass of genomicDNA before and after each roundof replication
  4. 4.  The Meselson-Stahlexperiment showed that thenitrogen used for thesynthesis of new dsDNAbecomes equally dividedbetween the two daughtergenomes This suggests asemiconservative replicationmechanism
  5. 5.  Both strands are replicated simultaneously
  6. 6.  Parental DNA strand servesas a template Nucleotide triphosphatesserve as substrates in strandsynthesis 3’ HydroxylPrimer - thegrowing end of the chainmakes a bond to the α-phosphorus of nucleotide Pyrophosphate is a goodleaving group – separatelyhydrolysed to Pi irreversible
  7. 7.  Polymerase I is most abundant but itsprimary function is in clean-up duringreplication, repair, and recombination Polymerase II is probably responsible forDNA repair Polymerase III is responsible for DNAreplication
  8. 8.  Initiation◦ Requires initiator proteins (trans-acting factors) Elongation◦ Leading and Lagging strands (repeated priming) Termination◦ Circular and linear chromosomes have unique problems
  9. 9.  DNA Primase Synthesizes Short RNA PrimerMolecules on the Lagging Strand Helicases - Open Up the DNA Double Helixin Front of the Replication Fork Single strand binding proteins keep ssDNAout of trouble Clamp subunits tether A Moving DNAPolymerase to the DNA The Proteins at a Replication ForkCooperate to Form a Replication Machine
  10. 10.  Chemical reactions and some physical processes constantlydamage genomic DNA◦ At the molecular level, damage usually involves changes in the structureof one of the strands◦ Vast majority are corrected by repair systems using the other strand as atemplate◦ Some base changes escape repair and the incorrect base serves as atemplate in replication◦ The daughter DNA carries a changed sequence in both strands; the DNAhas been mutated Accumulation of mutations in eukaryotic cells is stronglycorrelated with cancer; most carcinogens are also mutagens
  11. 11.  Mismatches arise from occasional incorporationof incorrect nucleotides Abnormal bases arise from spontaneousdeamination reactions or via chemical alkylation Pyrimidine dimers form when DNA is exposedto UV light Backbone lesions occur from exposure toionizing radiation
  12. 12. The fundamental difference betweenprokaryotes and eukaryotes is thatprokaryotes have a single type ofchromosome, while most eukaryotes have adiploid number of chromosomes of severaldifferent types in somatic cells
  13. 13. •The complete set of all metaphasechromosomes in a cell is called itskaryotype•Karyotypes are species specific, and cellsof organisms within the same species willhave the same karyotype
  14. 14. •Human karyotypes show chromosomesarranged in order according to size andposition of the centromere•Karyotypes allow geneticists to identifycertain chromosome mutations thatcorrelate with congenital abnormalities
  15. 15. Example of the human karyotypeExample of the human karyotype11 22 33 44 5566 77 88 99 1010 1111 12121313 1414 1515 1616 1717 18181919 2020 2121 2222 XX YY
  16. 16. • Certain regions called bands onchromosomes stain more intensely thatother regions• Banding patterns are specific for eachchromosome and allow the chromosomes tobe distinguished• G-banding produces bands onchromosomes when they are stained withGiemsa stainChromosomes are first heat treated orsubjected to proteolytic enzymes• G bands reflect regions of DNA rich in ATresidues (300 G bands distinguished in
  17. 17. Example of the human karyotypeExample of the human karyotype11 22 33 44 5566 77 88 99 1010 1111 12121313 1414 1515 1616 1717 18181919 2020 2121 2222 XX YY
  18. 18. • Q-banding produces bands whenchromosomes are stained with quinacrinedye, which binds preferentially to AT-richregions of DNA• In FISH (Fluorescence In SituHybridization), chromosomes are stainedwith fluorescent tags attached to specificDNA sequences• Purpose of banding pattern: cytogeneticanalysis and landmarks of locating genes(mapping genes)
  19. 19. Designations ofDesignations ofthe bands andthe bands andinterbands in theinterbands in thehuman karyotypehuman karyotypeG Banding Pattern
  20. 20. • An organism’s total DNA content is called itsC-value: total amount of DNA in a haploid cell• The amount of genetic material in a cell variesgreatly among prokaryotes and eukaryotes• A direct relationship does not exist betweenthe C value and the structural ororganizational complexity of the organism• One reason for this is the variation in theamount of repetitive DNA sequences in thegenome
  21. 21. No direct relationshipNo direct relationshipbetween the C value andbetween the C value andthe structural orthe structural ororganizationalorganizationalcomplexity of thecomplexity of theorganismorganism
  22. 22. • The large amount of DNA present ineukaryotic chromosomes is compacted byassociation with histones, formingstructures called nucleosomes• Nucleosomes fold further into chromatinfibers• Each chromosome contains a large numberof looped domains of 30-nm chromatinfibers attached to a protein scaffold
  23. 23. HISTONES•Small basic proteins•Constant amount in cells•25% lysine & arginine (Net + charge)•5 main types: H1, H2A, H2B, H3 & H4
  24. 24. •Equal amount of histones & DNA•H2A, H2B, H3 & H4 are highly conservedamong distinct species•Histone proteins are among the mostconserved proteins•H1 varies in cells (in RBC it is replaced byH5)
  25. 25. NON-HISTONE PROTEINS•All DNA chromosomal proteins minushistones•Structural proteins or enzymes i.e. DNAreplication enzymes, regulatory proteins,transcription factors…•Differ in number and type in different celltypes
  26. 26. • Acidic proteins (negatively charged)• Equal amount of non-histones & DNA• Example of HMGs (High-Motility Group proteins)Bind to minor grooveHave a role in DNA bendingHave a role in formation of higher orderchromatin structure
  27. 27. • Octamer of histones 2 (H2A, H2B, H3, H4)+ linker histone H1 + 180 bp of DNA• DNA compacts by winding 1 and ¾ turn ofthe outside of the histone octamer• Under electron microscopy, 11 nmchromatin fiber (beads on a string)
  28. 28. Nucleosome StructureNucleosome Structure
  29. 29. Nucleosomes connected together by linkerNucleosomes connected together by linkerDNA and H1 histone to produce the “beads-DNA and H1 histone to produce the “beads-on-a-string” extended form of chromatinon-a-string” extended form of chromatin
  30. 30. Packaging of nucleosomes into the 30-nmPackaging of nucleosomes into the 30-nmchromatin fiberchromatin fiber
  31. 31. The many different ordersThe many different ordersof chromatin packing thatof chromatin packing thatgive rise to the highlygive rise to the highlycondensed metaphasecondensed metaphasechromosome (700 Xchromosome (700 Xcompaction)compaction)
  32. 32. • The functional state of thechromosome is related to the extent ofcoiling• The more condensed areas of thechromosome (heterochromatin) aregenetically inactive• The less compacted regions(euchromatin) contain genes that areexpressed
  33. 33. • Centromeres are the sites at whichchromosomes attach to the mitotic andmeiotic spindles• Consensus yeast centromeric region8bp-78 to 86 bp >90%AT-25bp• The centromere region of each eukaryoticchromosome is responsible for accuratesegregation of the replicated chromosometo the progeny cells during both mitosisand meiosis
  34. 34. The CentromereThe Centromere
  35. 35. • Telomeres are regions found at the end ofchromosomes• They are often associated with the nuclearenvelope and are common tochromosomes of the same species• Telomeres are needed for chromosomestability
  36. 36. Centromere and TelomeresCentromere and Telomeres

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