Chapter 13Chapter 13

DNA and Its Role in HeredityDNA and Its Role in Heredity

Review of DNA StructureReview of DNA Structure

Four key features of DNA structure:Four key features of DNA structure:

• It is a It is a double-stranded helixdouble-stranded helix of uniform of uniform diameterdiameter

• It is It is right-handedright-handed

• It is It is antiparallelantiparallel

• Outer edges of nitrogenous bases are Outer edges of nitrogenous bases are exposedexposed in the major and minor in the major and minor groovesgrooves

Review of DNA StructureReview of DNA Structure

Complementary base pairing:Complementary base pairing:

• Adenine (A) pairs with thymine (T) by Adenine (A) pairs with thymine (T) by two hydrogen bondstwo hydrogen bonds

• Cytosine (C) pairs with guanine (G) by Cytosine (C) pairs with guanine (G) by three hydrogen bondsthree hydrogen bonds

• Every Every base pairbase pair consists of one purine consists of one purine and one pyrimidineand one pyrimidine

Review of DNA StructureReview of DNA Structure

Two Copies of the same sequence, just reverse compliments

Four Important Functions of DNAFour Important Functions of DNA

1) Stores genetic information1) Stores genetic information2) Is susceptible to mutation2) Is susceptible to mutation

3) 3) Is precisely replicated in cell Is precisely replicated in cell divisiondivision

4) Is expressed as the phenotype4) Is expressed as the phenotype

DNA Replication DNA Replication Three possible replication Three possible replication

patterns:patterns:• SemiconservativeSemiconservative:: Parent serves Parent serves

as a template and new as a template and new molecules have one old and one molecules have one old and one new strandnew strand

• ConservativeConservative:: Original helix only serves as a Original helix only serves as a templatetemplate

• Dispersive:Dispersive: Parent fragments serve as Parent fragments serve as templates, assembling old and templates, assembling old and new parts into moleculesnew parts into molecules

Semi-Conservative Semi-Conservative Replication Replication

Two steps in DNA replication:Two steps in DNA replication:

• The double helix is unwound, making The double helix is unwound, making two template strandstwo template strands

• New nucleotides are added to the new New nucleotides are added to the new strand at the 3strand at the 3′ end′ end and joined by and joined by phosphodiester linkages. Sequence is phosphodiester linkages. Sequence is determined by complementary base determined by complementary base pairingpairing

Strands Grow from the 3’ EndStrands Grow from the 3’ End

How does Replication InitiateHow does Replication Initiate

A large protein complex called the A large protein complex called the “replication complex” interacts with “replication complex” interacts with the template strands.the template strands.

All chromosomes have a region called All chromosomes have a region called origin of replication (origin of replication (oriori).).

Proteins in the replication complex bind Proteins in the replication complex bind to a DNA sequence in to a DNA sequence in oriori..

Main Components in DNA Main Components in DNA Replication Replication

PrimasePrimase synthesizes RNA primers to start synthesizes RNA primers to start replication replication

DNA polymerase (I and III)DNA polymerase (I and III) adds nucleotides to adds nucleotides to the 3the 3′′ end. end.

DNA helicaseDNA helicase uses energy from ATP hydrolysis uses energy from ATP hydrolysis to unwind the DNA.to unwind the DNA.

Single-strand binding proteinsSingle-strand binding proteins keep the strands keep the strands from getting back together.from getting back together.

DNA ligase DNA ligase “glues” together any gaps in the “glues” together any gaps in the newly synthesized sequencenewly synthesized sequence

So how does it work?So how does it work?HelicaseHelicase will unwind the DNA Strand will unwind the DNA Strand

SS-Binding ProteinsSS-Binding Proteins keep the strands apart keep the strands apart

PrimasePrimase hops on and lays down a 10-20bp RNA hops on and lays down a 10-20bp RNA primerprimer

DNA polymerase IIIDNA polymerase III recognizes the primers and recognizes the primers and continues to extend the growing strand by reading continues to extend the growing strand by reading the complimentary base pairsthe complimentary base pairs

DNA polymerase IDNA polymerase I hopes on and replaces RNA hopes on and replaces RNA primer with DNA and proofreads the new sequenceprimer with DNA and proofreads the new sequence

DNA LigaseDNA Ligase then glues together any gaps then glues together any gaps

DNA PolymeraseDNA Polymerase

• Proof Reading Proof Reading AbilityAbility

• Can NOT start Can NOT start without a primer or without a primer or existing template existing template sequencesequence

Not all DNA Strands are EqualNot all DNA Strands are Equal

• Leading Strand Leading Strand “Easy Replication”“Easy Replication”

• Lagging Strand is Lagging Strand is slower and more slower and more difficultdifficult

• Replication ForkReplication Fork

Leading StrandLeading Strand

• Primer is created on Primer is created on the 3’ end of the the 3’ end of the existing sequenceexisting sequence

• Replication occurs Replication occurs following the following the Replication fork Replication fork until it falls off the until it falls off the other endother end

Lagging StrandLagging Strand

• Primer is created near Primer is created near the replication forkthe replication fork

• The strand is then The strand is then replicated moving away replicated moving away from the Replication from the Replication forkfork

• Forms Okazaki Forms Okazaki Fragments Fragments

After Replication…After Replication…

• DNA pol I replaces all DNA pol I replaces all RNA primers with DNARNA primers with DNA

• DNA ligase glues all DNA ligase glues all Okazaki Fragments Okazaki Fragments together together

• DNA pol can check for DNA pol can check for errors in sequencing errors in sequencing (proofreading)(proofreading)

PCRPCRCopies of DNA sequences can be made by Copies of DNA sequences can be made by

the polymerase chain reaction (PCR) the polymerase chain reaction (PCR) technique.technique.

PCR is a cyclical process:PCR is a cyclical process:• DNA fragments are denatured by heatingDNA fragments are denatured by heating• Primers, plus dNTPs and DNA Primers, plus dNTPs and DNA

polymerase are addedpolymerase are added• New DNA strands are synthesizedNew DNA strands are synthesized

PCRPCR

PCRPCR

PCR results in many copies of the DNA PCR results in many copies of the DNA fragment—referred to as fragment—referred to as amplifyingamplifying the the sequence.sequence.

The base sequence at the 3The base sequence at the 3′ end of the ′ end of the DNA fragment must be known.DNA fragment must be known.

Complementary primers, about 15Complementary primers, about 15––30 30 bases long, are made in the laboratory. bases long, are made in the laboratory.

PCRPCR

An initial problem with PCR was its An initial problem with PCR was its temperature requirements.temperature requirements.

The heat needed to denature the DNA The heat needed to denature the DNA destroyed most DNA polymerases.destroyed most DNA polymerases.

A DNA polymerase that does not A DNA polymerase that does not denature at high temperatures (90denature at high temperatures (90°°C) C) was taken from a hot springs was taken from a hot springs bacterium, bacterium, Thermus aquaticusThermus aquaticus..

Steps in a typical PCR CycleSteps in a typical PCR Cycle

1) Denature1) Denature

2) Annealing of the Primers2) Annealing of the Primers

3) Elongation of new sequence DNA Pol3) Elongation of new sequence DNA Pol

Leads to exponential amplification of Leads to exponential amplification of your target…your target…