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