DNA Replication Lecture - WordPress.com...DNA Replication D. DNA Primers DNA polymerases cannot initiate synthesis of a polynucleotide because they can only add nucleotides to the

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�� Chapter 16~Chapter 16~The Molecular Basis of InheritanceThe Molecular Basis of Inheritance

DNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA Structure

�� ChargaffChargaffratio of nucleotide ratio of nucleotide bases (A=T; C=G)bases (A=T; C=G)

�� Watson & Crick Watson & Crick (Wilkins, Franklin)(Wilkins, Franklin)

�� The Double Helix The Double Helix √√ nucleotidesnucleotides: :

nitrogenous base (thymine, nitrogenous base (thymine, adenine, cytosine, guanine); adenine, cytosine, guanine); sugar deoxyribose; sugar deoxyribose; phosphate groupphosphate group

DNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA StructureDNA Structure DNA BondingDNA BondingDNA BondingDNA BondingDNA BondingDNA BondingDNA BondingDNA Bonding

�� Purines: Purines: ‘‘AA’’ & & ‘‘GG’’

�� Pyrimidines: Pyrimidines: ‘‘CC’’ & & ‘‘TT’’(Chargaff rules)(Chargaff rules)

�� ‘‘AA’’ H+ bonds (2) with H+ bonds (2) with ‘‘TT’’and and ‘‘CC’’ H+ bonds (3) H+ bonds (3) with with ‘‘GG’’

�� Van der Waals Van der Waals attractions between the attractions between the stacked pairsstacked pairs

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DNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTUREDNA STRUCTURE 1. DNA Replication1. DNA Replication1. DNA Replication1. DNA Replication1. DNA Replication1. DNA Replication1. DNA Replication1. DNA Replication

A. OverviewA. OverviewA. OverviewA. OverviewA. OverviewA. OverviewA. OverviewA. Overview�� In 2In 2ndnd paper Watson and Crick published hypothesis for how paper Watson and Crick published hypothesis for how

DNA replicates.DNA replicates.

�� Because each strand is complementary, each can form a Because each strand is complementary, each can form a template when separated.template when separated.

�� When a cell copies a DNA molecule, each strand serves as a When a cell copies a DNA molecule, each strand serves as a templatetemplate for ordering nucleotides into a new complementary for ordering nucleotides into a new complementary strand.strand.

�� Nucleotides line up along the template strand according to the Nucleotides line up along the template strand according to the basebase--pairing rulespairing rules..

�� The nucleotides are linked to form new strands.The nucleotides are linked to form new strands.

DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: B. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA Replication

�� Watson and Crick: Watson and Crick: semiconservativesemiconservative replication, a replication, a double helix replicates, each of double helix replicates, each of the daughter molecules will have the daughter molecules will have 1 old strand and 1 new strand.1 old strand and 1 new strand.

�� Other models: Other models: conservativeconservative and and dispersivedispersive

�� MeselsonMeselson and Stahl supported and Stahl supported the the semiconservativesemiconservative modelmodel, , proposed by Watson and Crick, proposed by Watson and Crick, over the other two models:over the other two models:

DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: DNA Replication: B. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA Replication

1.1. Labeled the nucleotides of the old strands with Labeled the nucleotides of the old strands with a heavy isotope of nitrogen (15N), while any a heavy isotope of nitrogen (15N), while any new nucleotides were indicated by a lighter new nucleotides were indicated by a lighter isotope (14N).isotope (14N).

2.2. Replicated strands could be separated by Replicated strands could be separated by density in a centrifuge.density in a centrifuge.

3.3. Each modelEach model—— the semithe semi--conservative model, conservative model, the conservative model, and the dispersive the conservative model, and the dispersive modelmodel—— made specific predictions made specific predictions on the on the density of replicated DNA strandsdensity of replicated DNA strands..

4.4. The first replication in the 14N medium The first replication in the 14N medium produced a band of hybrid (15Nproduced a band of hybrid (15N--14N) DNA, 14N) DNA, eliminating the conservative modeleliminating the conservative model..

5.5. A second replication produced both light and A second replication produced both light and hybrid DNA, eliminating the dispersive model hybrid DNA, eliminating the dispersive model and supporting the and supporting the semiconservativesemiconservative model.model.

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DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication:DNA Replication:B. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA ReplicationB. Models for DNA Replication

DNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA ReplicationDNA Replication

2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication2. DNA Replication: A. Origins of Replication

In bacteriaIn bacteria, this is a single specific sequence of nucleotides that is reco, this is a single specific sequence of nucleotides that is recognized by the gnized by the replication enzymes.replication enzymes.

�� These enzymes separate the strands, forming a These enzymes separate the strands, forming a replication replication ““bubblebubble””..

In eukaryotesIn eukaryotes, there may be hundreds or thousands of origin sites per chromos, there may be hundreds or thousands of origin sites per chromosomeome

�� HelicaseHelicase is the enzyme that catalyzes the untwisting of DNA at the repliis the enzyme that catalyzes the untwisting of DNA at the replication forkcation fork

�� Replication forksReplication forks: the Y: the Y--shaped region where new DNA is elongating.shaped region where new DNA is elongating.

�� The replication bubbles elongate as the DNA is replicated and evThe replication bubbles elongate as the DNA is replicated and eventually fuse entually fuse

2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication2. DNA Replication

B. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNA

�� DNADNA polymerasespolymerases catalyze the elongation of catalyze the elongation of new DNA at a replication fork.new DNA at a replication fork.

�� As nucleotides align with complementary As nucleotides align with complementary bases along the template strand, they are bases along the template strand, they are added to the growing end of the new strand by added to the growing end of the new strand by the polymerase.the polymerase.

�� The rate of elongation is about 500 nucleotides The rate of elongation is about 500 nucleotides per second in bacteria and per second in bacteria and 50 per second50 per second in in human cells. human cells.

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B. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNAB. Elongation of New DNA

�� Raw nucleotides = nucleoside Raw nucleotides = nucleoside triphosphatestriphosphates: : nitrogen base, nitrogen base, deoxyribosedeoxyribose, , triphosphatetriphosphate tail.tail.

�� As each nucleotide is added, the last two As each nucleotide is added, the last two phosphate groups are hydrolyzed to form phosphate groups are hydrolyzed to form pyrophosphatepyrophosphate..

�� The The exergonicexergonic hydrolysis hydrolysis of pyrophosphate to of pyrophosphate to two two inorganic phosphateinorganic phosphatemolecules drives molecules drives the polymerization of the polymerization of the nucleotide to the the nucleotide to the new strand.new strand.


C. C. C. C. C. C. C. C. AntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallel Nature of DNANature of DNANature of DNANature of DNANature of DNANature of DNANature of DNANature of DNA

�� The sugarThe sugar--phosphate backbones of phosphate backbones of the double helix run in opposite the double helix run in opposite directions.directions.

�� Each DNA strand: Each DNA strand: 33’’ endend: free hydroxyl group attached : free hydroxyl group attached

to to deoxyribosedeoxyribose55’’ endend: free phosphate group : free phosphate group

attached to attached to deoxyribosedeoxyribose. . �� The 5The 5’’ --> 3> 3’’ direction of one strand direction of one strand

runs counter to the 3runs counter to the 3’’ --> 5> 5’’ direction direction of the other strand.of the other strand.

�� DNA polymerases can only add DNA polymerases can only add nucleotides to the free 3nucleotides to the free 3’’ end of a end of a growing DNA strand.growing DNA strand.

�� A new DNA strand can only A new DNA strand can only elongate in the 5elongate in the 5’’-->3>3’’ direction direction

��(1(1’’--55’’ refer to refer to numbers of numbers of carbons on carbons on deoxyribosedeoxyribose sugar)sugar)

2. DNA Replication:2. DNA Replication:2. DNA Replication:2. DNA Replication:2. DNA Replication:2. DNA Replication:2. DNA Replication:2. DNA Replication:

C. C. C. C. C. C. C. C. AntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallelAntiparallel Nature of DNANature of DNANature of DNANature of DNANature of DNANature of DNANature of DNANature of DNA

�� This creates a problem at the replication fork This creates a problem at the replication fork because one parental strand is oriented because one parental strand is oriented 33’’-->5>5’’into the fork, while the other into the fork, while the other antiparallelantiparallel parental parental strand is oriented strand is oriented 55’’-->3>3’’ into the fork.into the fork.

�� At the replication fork, one parental strand (3At the replication fork, one parental strand (3’’--> > 55’’ into the fork), the into the fork), the leading strandleading strand, can be , can be used by polymerases as a template for a used by polymerases as a template for a continuous complementary strandcontinuous complementary strand..

�� The other parental strand (5The other parental strand (5’’-->3>3’’ into the fork), into the fork), the the lagging strandlagging strand, is copied away from the , is copied away from the fork in short segments (Okazaki fragments).fork in short segments (Okazaki fragments).

�� Okazaki fragmentsOkazaki fragments, each about 100, each about 100--200 200 nucleotides, are joined by nucleotides, are joined by DNA DNA ligaseligase to form to form the sugarthe sugar--phosphate backbone of a single DNA phosphate backbone of a single DNA strand.strand.

DNA Replication: the leading DNA Replication: the leading DNA Replication: the leading DNA Replication: the leading DNA Replication: the leading DNA Replication: the leading DNA Replication: the leading DNA Replication: the leading

strandstrandstrandstrandstrandstrandstrandstrand

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DNA Replication: the lagging DNA Replication: the lagging DNA Replication: the lagging DNA Replication: the lagging DNA Replication: the lagging DNA Replication: the lagging DNA Replication: the lagging DNA Replication: the lagging

strandstrandstrandstrandstrandstrandstrandstrand


D. DNA PrimersD. DNA PrimersD. DNA PrimersD. DNA PrimersD. DNA PrimersD. DNA PrimersD. DNA PrimersD. DNA Primers

�� DNA polymerases cannot DNA polymerases cannot initiateinitiate synthesis of a synthesis of a polynucleotide because they can only polynucleotide because they can only add nucleotides add nucleotides to the end of an existing chainto the end of an existing chain that is basethat is base--paired with paired with the template strand.the template strand.

�� To start a new chain requires a To start a new chain requires a primerprimer, a short , a short segment of RNA: about 10 segment of RNA: about 10 nulceotidesnulceotides longlong

�� PrimasePrimase, an RNA polymerase, links , an RNA polymerase, links ribonucleotidesribonucleotidesthat are complementary to the DNA template into the that are complementary to the DNA template into the primer.primer.

�� RNA polymerases can start an RNA chain from a RNA polymerases can start an RNA chain from a single template strand.single template strand.

�� After formation of the primer, DNA polymerases can After formation of the primer, DNA polymerases can add add deoxyribonucleotidesdeoxyribonucleotides to the 3to the 3’’ end of the end of the ribonucleotideribonucleotide chain.chain.

2. DNA Replication D. Primers2. DNA Replication D. Primers2. DNA Replication D. Primers2. DNA Replication D. Primers2. DNA Replication D. Primers2. DNA Replication D. Primers2. DNA Replication D. Primers2. DNA Replication D. Primers�� Another DNA polymerase later Another DNA polymerase later

replaces the primer replaces the primer ribonucleotidesribonucleotideswith with deoxyribonucleotidesdeoxyribonucleotidescomplementary to the template.complementary to the template.

�� The leading strand requires the The leading strand requires the formation of only a single primer as formation of only a single primer as the replication fork continues to the replication fork continues to separate.separate.

�� The lagging strand requires The lagging strand requires formation of a new primer as the formation of a new primer as the replication fork progresses.replication fork progresses.

�� After the primer is formed, DNA After the primer is formed, DNA polymerase can add new polymerase can add new nucleotides away from the fork until nucleotides away from the fork until it runs into the previous Okazaki it runs into the previous Okazaki fragment. fragment.

�� The primers are converted to DNA The primers are converted to DNA before DNA before DNA ligaseligase joins the joins the fragments together.fragments together.


E. Summary of Enzymes InvolvedE. Summary of Enzymes InvolvedE. Summary of Enzymes InvolvedE. Summary of Enzymes InvolvedE. Summary of Enzymes InvolvedE. Summary of Enzymes InvolvedE. Summary of Enzymes InvolvedE. Summary of Enzymes Involved

DNA polymerase molecules DON’T move along a stationary DNA template!

The DNA polymerase molecules “reel in” the parental DNA and “extrude”newly made daughter DNA molecules.

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3. 3. 3. 3. 3. 3. 3. 3. ProfreadingProfreadingProfreadingProfreadingProfreadingProfreadingProfreadingProfreading DNADNADNADNADNADNADNADNA

�� Mismatched nucleotides that Mismatched nucleotides that are missed by DNA are missed by DNA polymerase or mutations that polymerase or mutations that occur after DNA synthesis is occur after DNA synthesis is completed can often be completed can often be repaired: over 130 repair repaired: over 130 repair enzymes identified in enzymes identified in humans!humans!

Mismatch repair, special enzymes fix incorrectly paired nucleotides.

A hereditary defect in one of these enzymes is associated with a form of colon cancer

Nucleotide excision repair, a nuclease cuts out a damaged strand.Gap is filled in by DNA polymerase and ligase.The importance of the proper functioning of repair enzymes: xeroderma pigmentosum.

3. 3. 3. 3. 3. 3. 3. 3. ProfreadingProfreadingProfreadingProfreadingProfreadingProfreadingProfreadingProfreading DNA DNA DNA DNA DNA DNA DNA DNA --------

MutationMutationMutationMutationMutationMutationMutationMutation

4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends�� Limitations in the DNA polymerase: Limitations in the DNA polymerase:

problems for the linear DNA of problems for the linear DNA of eukaryotic chromosomes.eukaryotic chromosomes.

�� No way to complete the 5No way to complete the 5’’ ends of ends of daughter DNA strands.daughter DNA strands.

�� Repeated rounds of replication Repeated rounds of replication produce shorter and shorter DNA produce shorter and shorter DNA molecules.molecules.

�� The ends of eukaryotic The ends of eukaryotic chromosomal DNA molecules, the chromosomal DNA molecules, the telomerestelomeres, have special nucleotide , have special nucleotide sequences.sequences.

�� In human telomeres, this sequence In human telomeres, this sequence is typically TTAGGG, repeated is typically TTAGGG, repeated between 100 and 1,000 times.between 100 and 1,000 times.

�� Telomeres protect genes from Telomeres protect genes from being eroded through multiple being eroded through multiple rounds of DNA replication.rounds of DNA replication.

�� Eukaryotic cells have evolved a Eukaryotic cells have evolved a mechanism to restore shortened mechanism to restore shortened telomeres.telomeres.

4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends�� TelomeraseTelomerase uses a short molecule of uses a short molecule of

RNA as a template to extends the 3RNA as a template to extends the 3’’end of the telomere.end of the telomere.

�� PrimasePrimase and DNA polymerase now and DNA polymerase now extend the 5extend the 5’’ end.end.

�� It does not repair the 3It does not repair the 3’’--end end ““overhang,overhang,””butbut it does lengthen the it does lengthen the telomere.telomere.

�� Telomerase is Telomerase is not not present in most present in most cells of cells of multicellularmulticellular organisms.organisms.

�� DNA of dividing somatic cells and DNA of dividing somatic cells and cultured cells does tend to become cultured cells does tend to become shorter.shorter.

�� Telomere lengthTelomere length: a limiting factor in : a limiting factor in the life span of certain tissues and of the life span of certain tissues and of the organism.the organism.

�� TelomeraseTelomerase: in germ: in germ--line cells, line cells, ensuring that zygotes have long ensuring that zygotes have long telomeres.telomeres.

�� Active telomerase is also found in Active telomerase is also found in cancerous somatic cells.cancerous somatic cells.

�� This overcomes the progressive This overcomes the progressive shortening that would eventually lead shortening that would eventually lead to selfto self--destruction of the cancer destruction of the cancer

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4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA ends4. Replication of DNA endsDNA DNA DNA DNA DNA DNA DNA DNA

REPLICATIONREPLICATIONREPLICATIONREPLICATIONREPLICATIONREPLICATIONREPLICATIONREPLICATION

ENZYMES:ENZYMES:ENZYMES:ENZYMES:ENZYMES:ENZYMES:ENZYMES:ENZYMES:

DNA Replication Lecture - WordPress.com...DNA Replication D. DNA Primers DNA polymerases cannot initiate synthesis of a polynucleotide because they can only add nucleotides to the

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