chapter 5 & 6 dna & dna replication. history dna dna comprised of genes in non-dividing cell...
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Chapter 5 & 6Chapter 5 & 6
DNA & DNA ReplicationDNA & DNA Replication
HistoryHistoryDNADNA
Comprised of genesComprised of genes In non-dividing cell nucleus In non-dividing cell nucleus
as chromatinas chromatinProtein/DNA complexProtein/DNA complex
Chromosomes form during Chromosomes form during cell divisioncell divisionDuplicate to yield a full set in Duplicate to yield a full set in
daughter celldaughter cell
DNA is Genetic MaterialDNA is Genetic Material
From Chapter 2From Chapter 2Nucleic acids are polymersNucleic acids are polymers
Monomers are called nucleotidesMonomers are called nucleotidesNucleotides = base + sugar + phosphateNucleotides = base + sugar + phosphate
Base = purine or pyrimidineBase = purine or pyrimidine Purines = adenine, guaninePurines = adenine, guanine Pyrimidines = thymine, cytosine, uracilPyrimidines = thymine, cytosine, uracil
Sugar = deoxyribose or riboseSugar = deoxyribose or ribosePhosphate, a single phosphate in DNAPhosphate, a single phosphate in DNA
Sugar of nt 1 is linked to the phosphate of nt Sugar of nt 1 is linked to the phosphate of nt 2 by a phosphodiester bond2 by a phosphodiester bond
Panel 2-6Panel 2-6
Chapter 2 Chapter 2 – cont’d– cont’d
DNA is a Double HelixDNA is a Double Helix NucleotidesNucleotides
A, G, T, CA, G, T, C
Sugar and phosphate Sugar and phosphate form the backboneform the backbone
Bases lie between the Bases lie between the backbonebackbone
Held together by Held together by H-bonds between the H-bonds between the basesbases A-T – 2 H bondsA-T – 2 H bonds G-C – 3 H bondsG-C – 3 H bonds
H - BondsH - Bonds Base-pairing rulesBase-pairing rules AAT only (AT only (AU if DNA-RNA U if DNA-RNA
hybrid)hybrid) GGC onlyC only
DNA strand has DNA strand has directionality – one end is directionality – one end is different from the other enddifferent from the other end
2 strands are anti-parallel, 2 strands are anti-parallel, run in opposite directionsrun in opposite directions Complementarity resultsComplementarity results Important to replicationImportant to replication
Helical StructureHelical Structure
Nucleotides as LanguageNucleotides as Language
We must start to think of the nucleotides – We must start to think of the nucleotides – A, G, C and T as part of a special A, G, C and T as part of a special language – the language of genes that we language – the language of genes that we will see translated to the language of will see translated to the language of amino acids in proteinsamino acids in proteins
Genes as Information TransferGenes as Information Transfer
A A genegene is the sequence of nucleotides is the sequence of nucleotides within a portion of DNA that codes for a within a portion of DNA that codes for a peptide or a functional RNApeptide or a functional RNA
Sum of all genes = Sum of all genes = genomegenome
DNA ReplicationDNA Replication
SemiconservativeSemiconservative Daughter DNA is a Daughter DNA is a
double helix with 1 double helix with 1 parent strand and 1 parent strand and 1 new strandnew strand
Found that 1 strand Found that 1 strand serves as the serves as the templatetemplate for new for new strandstrand
DNA TemplateDNA Template
Each strand of the parent DNA is used as a Each strand of the parent DNA is used as a templatetemplate to make the new daughter strand to make the new daughter strand
DNA replication makes 2 new complete double DNA replication makes 2 new complete double helices each with 1 old and 1 new strandhelices each with 1 old and 1 new strand
Replication OriginReplication Origin
Site where replication Site where replication begins begins 1 in E. coli1 in E. coli 1,000s in human1,000s in human
Strands are separated to Strands are separated to allow replication machinery allow replication machinery contact with the DNAcontact with the DNA Many A-T base pairs Many A-T base pairs
because easier to break 2 because easier to break 2 H-bonds that 3 H-bondsH-bonds that 3 H-bonds
Note anti-parallel chainsNote anti-parallel chains
Replication ForkReplication Fork
Bidirectional movement of the DNA replication machineryBidirectional movement of the DNA replication machinery
DNA PolymeraseDNA Polymerase An enzyme that An enzyme that
catalyzes the addition of catalyzes the addition of a nucleotide to the a nucleotide to the growing DNA chain growing DNA chain
Nucleotide enters as a Nucleotide enters as a nucleotide tri-POnucleotide tri-PO44
3’–OH of sugar attacks 3’–OH of sugar attacks first phosphate of tri-first phosphate of tri-POPO44 bond on the 5’ C of bond on the 5’ C of
the new nucleotidethe new nucleotide releasing pyrophosphate releasing pyrophosphate
(PP(PPii) + energy) + energy
DNA PolymeraseDNA Polymerase
Bidirectional synthesis of the DNA double Bidirectional synthesis of the DNA double helixhelix
Corrects mistaken base pairingsCorrects mistaken base pairingsRequires an established polymer (small Requires an established polymer (small
RNA RNA primerprimer) before addition of more ) before addition of more nucleotidesnucleotides
Other proteins and enzymes necessaryOther proteins and enzymes necessary
How is DNA Synthesized?How is DNA Synthesized?
Original theory Original theory Begin adding nucleotides at originBegin adding nucleotides at origin Add subsequent bases following pairing rulesAdd subsequent bases following pairing rules
Expect both strands to be synthesized simultaneouslyExpect both strands to be synthesized simultaneously This is NOT how it is accomplishedThis is NOT how it is accomplished
Why DNA Why DNA Isn’t Isn’t
Synthesized Synthesized 3’3’5’5’
Correction: Refer to Figure 6-15 on page 205 of your textbook for “corrected” figure. This figure fails to show the two terminal phosphate groups attached on the 5’ end of the nucleotide strand located at the top of this figure.
How is DNA Synthesized?How is DNA Synthesized?
Actually how DNA is synthesizedActually how DNA is synthesizedSimple addition of nucleotides along one Simple addition of nucleotides along one
strand, as expectedstrand, as expectedCalled the Called the leading strandleading strand DNA polymerase reads 3’ DNA polymerase reads 3’ 5’ along the leading 5’ along the leading
strand from the RNA primerstrand from the RNA primerSynthesis proceeds 5’ Synthesis proceeds 5’ 3’ with respect to the 3’ with respect to the
new daughter strandnew daughter strand
Remember how the nucleotides are Remember how the nucleotides are added!!!!! added!!!!! 5’ 5’ 3’ 3’
How is DNA Synthesized?How is DNA Synthesized?
Actually how DNA is synthesizedActually how DNA is synthesizedOther daughter strand is also synthesized Other daughter strand is also synthesized
5’5’3’ because that is only way that DNA can 3’ because that is only way that DNA can be assembledbe assembled
However the template is also being read However the template is also being read 5’5’3’3’Compensate for this by feeding the DNA strand Compensate for this by feeding the DNA strand
through the polymerase, and primers and make through the polymerase, and primers and make many short segments that are later joined (ligated) many short segments that are later joined (ligated) togethertogether
Called the Called the lagging strandlagging strand
DNA Replication Fork Fig 6-12DNA Replication Fork Fig 6-12
Mistakes during ReplicationMistakes during Replication Base pairing rules must be maintainedBase pairing rules must be maintained
Mistake = genome mutation, may have Mistake = genome mutation, may have consequence on daughter cellsconsequence on daughter cells
Only correct pairings fit in the polymerase Only correct pairings fit in the polymerase active siteactive site
If wrong nucleotide is includedIf wrong nucleotide is included Polymerase uses its Polymerase uses its proofreadingproofreading ability to cleave ability to cleave
the phosphodiester bond of improper nucleotidethe phosphodiester bond of improper nucleotide Activity 3’ Activity 3’ 5’ 5’
And then adds correct nucleotide and proceeds And then adds correct nucleotide and proceeds down the chain again in the 5’ down the chain again in the 5’ 3’ direction 3’ direction
ProofreadingProofreading
Starting SynthesisStarting Synthesis
DNA polymerase can only ADD nucleotides DNA polymerase can only ADD nucleotides to a growing polymerto a growing polymer
Another enzyme, Another enzyme, primaseprimase, synthesizes a , synthesizes a short RNA chain called a short RNA chain called a primerprimerDNA/RNA hybrid for this short stretchDNA/RNA hybrid for this short stretchBase pairing rules followed (BUT A-U)Base pairing rules followed (BUT A-U)Later removed, replaced by DNA and the Later removed, replaced by DNA and the
backbone is sealed (ligated)backbone is sealed (ligated)
Primers – cont’dPrimers – cont’d Simple addition of primer Simple addition of primer
along leading strandalong leading strand RNA primer synthesized 5’ RNA primer synthesized 5’
3’, then polymerization 3’, then polymerization with DNAwith DNA
Many primers are needed Many primers are needed along the lagging strandalong the lagging strand 1 primer per small 1 primer per small
fragment of new DNA fragment of new DNA made along the lagging made along the lagging strand strand
Called Called Okazaki fragmentsOkazaki fragments
Removal of PrimersRemoval of Primers
Other enzymes needed to excise Other enzymes needed to excise (remove) the primers(remove) the primersNucleaseNuclease – removes the RNA primer – removes the RNA primer
nucleotide by nucleotidenucleotide by nucleotideRepair polymeraseRepair polymerase – replaces RNA with DNA – replaces RNA with DNADNA ligaseDNA ligase – seals the sugar-phosphate – seals the sugar-phosphate
backbone by creating phosphodiester bondbackbone by creating phosphodiester bondRequires MgRequires Mg2+2+ and ATP and ATP
Other Necessary ProteinsOther Necessary Proteins
HelicaseHelicase opens double helix and helps it opens double helix and helps it uncoiluncoil
Single-strand binding proteinsSingle-strand binding proteins (SSBP) keep (SSBP) keep strands separated – large amount of this strands separated – large amount of this protein requiredprotein required
Sliding clampSliding clamp Subunit of polymeraseSubunit of polymerase Helps polymerase slide along strandHelps polymerase slide along strand
All are coordinated with one another to All are coordinated with one another to produce the growing DNA strand (protein produce the growing DNA strand (protein machine)machine)
Components of the DNA ReplicationComponents of the DNA Replication
Polymerase & Proteins CoordinatedPolymerase & Proteins Coordinated
One polymerase complex apparently synthesizes One polymerase complex apparently synthesizes leading/lagging strands simultaneouslyleading/lagging strands simultaneously
Even more complicated in eukaryotesEven more complicated in eukaryotes
DNA RepairDNA RepairFor the rare mutations occurring during For the rare mutations occurring during
replication that isn’t caught by DNA replication that isn’t caught by DNA polymerase proofreadingpolymerase proofreading
For mutations occurring with daily For mutations occurring with daily assaultassault
If no repairIf no repair In germ (sex) cells In germ (sex) cells inherited diseases inherited diseases In somatic (regular) cells In somatic (regular) cells cancer cancer
Effect of MutationEffect of Mutation
Uncorrected Replication ErrorsUncorrected Replication Errors
Mismatch repairMismatch repair Enzyme complex recognizes mistake and excises Enzyme complex recognizes mistake and excises
newly-synthesized strand and fills in the correct newly-synthesized strand and fills in the correct pairingpairing
Mismatch Repair – cont’dMismatch Repair – cont’d
Eukaryotes “label” Eukaryotes “label” the daughter strand the daughter strand with nicks to with nicks to recognize the new recognize the new strandstrand Separates new from Separates new from
oldold
Depurination or DeaminationDepurination or Deamination
DepurinationDepurination – removal of a purine base from – removal of a purine base from the DNA strandthe DNA strand
DeaminationDeamination is the removal of an amine group is the removal of an amine group on Cytosine to yield Uracilon Cytosine to yield Uracil Could lead to the insertion of Adenine rather than Could lead to the insertion of Adenine rather than
Guanosine on next roundGuanosine on next round
Chemical ModificationsChemical Modifications
Thymine DimersThymine Dimers
Caused by exposure to UV lightCaused by exposure to UV light 2 adjacent thymine residues become 2 adjacent thymine residues become
covalently linkedcovalently linked
Repair Repair MechanismsMechanisms
Different enzymes Different enzymes recognize, excise recognize, excise different mistakesdifferent mistakes
DNA polymerase DNA polymerase synthesizes proper synthesizes proper strandstrand
DNA ligase joins new DNA ligase joins new fragment with the fragment with the polymerpolymer