Chapter 16:

DNA Structure & Replication

2. DNA Replication

1. DNA Structure

1. DNA Structure

Chapter Reading – pp. 313-318

Genetic Material: Protein or DNA?

Until the early 1950’s no one knew for sure, but it

was generally thought that protein was the genetic

material. Why?

• protein is made of 20 different amino acids

• DNA is made of only 4 different nucleotides

• protein could theoretically store more info

• a “20 letter alphabet” vs a “4 letter alphabet”

• it was assumed that life was so complex, therefore a

“bigger alphabet” was necessary to somehow encode it!

Some classic experiments would prove otherwise…

Living S cells(control)

Living R cells(control)

Heat-killedS cells(control)

Mixture ofheat-killedS cells andliving R cells

Mouse dies Mouse diesMouse healthy Mouse healthy

Living S cells

EXPERIMENT

RESULTS

Transformation of Bacteria

(Frederick Griffith, 1928)

Demonstrated

the transfer of

a genetic trait

between

different

bacteria.

The nature of

that genetic

material was

still unknown.

Phage

head

Tail

sheath

Tail fiber

DNA

Bacterial

cell

100 n

m

What is the Genetic Material of

Bacteriophages?

Bacteriophages are viruses that infect bacteria.

• consist of a protein capsid which contains DNA

What enters the

bacterial host

cell, viral protein,

DNA, or both?

• whatever enters

the host cell

should be the

genetic material

Bacterial cell

Phage

Batch 1:Radioactivesulfur(35S)

Radioactiveprotein

DNA

Batch 2:Radioactivephosphorus(32P)

RadioactiveDNA

Emptyproteinshell

PhageDNA

Centrifuge

Centrifuge

Radioactivity(phage protein)in liquid

Pellet (bacterialcells and contents)

PelletRadioactivity(phage DNA)in pellet

EXPERIMENT

Bacteriophage Genetic Material is DNA

(Alfred Hershey & Martha Chase, 1952)

(a) Rosalind Franklin (b) Franklin’s X-ray diffractionphotograph of DNA

The Discovery of DNA Structure

Using the technique of x-ray crystallography,

Rosalind Franklin, James Watson & Francis Crick

figured out the structure of DNA

• Watson & Crick used the X-ray diffraction data of Rosalind

Franklin to deduce the structure of DNA

DNA: a Polymer of 4 Nucleotides

T

GC

A

pyrimidines purines

Sugar–phosphatebackbone

Nitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

Nitrogenous base

Phosphate

DNA nucleotide

Sugar(deoxyribose)

3 end

5 endStructure of a

DNA Strand

A single DNA polymer

or “strand” consists of

a sugar-phosphate

backbone with the

bases project out.

The ends of a DNA

strand are different,

with one end having a

free 5’ phosphate, and

the other having a free

3’ hydroxyl group

3.4 nm

1 nm

0.34 nm

Hydrogen bond

(a) Key features ofDNA structure

Space-fillingmodel

(c)(b) Partial chemical structure

3 end

5 end

3 end

5 end

T

T

A

A

G

G

C

C

C

C

C

C

C

C

C

C

C

G

G

G

G

G

G

G

G

G

T

T

T

T

T

T

A

A

A

A

A

A

Structure of Double-stranded DNA• the 2 strands are anti-parallel and interact via base pairs

Purine purine: too wide

Pyrimidine pyrimidine: too narrow

Purine pyrimidine: widthconsistent with X-ray data

Sugar

Sugar

Sugar

Sugar

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

DNA “Base-Pairing”Base pairs are held together by hydrogen bonds.

Why only A:T and C:G?

• the position of chemical

groups involved in H-Bonds

• the size of the bases

(purine & pyrimidine)

The DNA “Sequence”

The DNA sequence is the linear order of

nucleotides in a DNA strand:

• each DNA strand in the double helix has its own

sequence

• the sequences in each strand are considered as

complementary to each other

e.g.

5’ A–C–A–C–A–C–A–C–A–C 3’3’ T–G–T–G–T–G–T–G–T–G 5’

• they differ, but “fit just right” with each other

• ea strand will “fit” with only 1 complementary strand

2. DNA Replication

Chapter Reading – pp. 318-330

How is DNA Replicated?

Every time a cell reproduces (i.e., divides) it

must replicate its chromosomes (DNA) during

S phase.

The process of DNA replication was originally

proposed to depend on the rules of base pairing:

• the sequence of one strand dictates the

sequence of the other

• each strand of the double helix could serve as a

template to make a complementary strand

• A:T & T:A , C:G & G:C

(a) Parent molecule (b) Separation ofstrands

(c) “Daughter” DNA molecules,each consisting of oneparental strand and onenew strand

A

A

A

A

A

A

A

A

A

A

A

A

T

T

T

T

T

T

T

T

T

T

T

T

C

C

C

C

C

C

C

C

G

G

G

G

G

G

G

G

The semiconservative model of DNA replication

proposed that each original strand serves as a template

to produce a new complementary strand.

• note that ea original strand ends up in a different molecule

Model for DNA Replication

(a) Conservativemodel

(b) Semiconservativemodel

(c) Dispersive model

Parentcell

Firstreplication

Secondreplication Other

Models

CONSERVATIVEThe original DNA

strands stay together

SEMICONSERVATIVEThe original DNA

strands remain intact

in separate molecules

DISPERSIVEThe original DNA

strands are dispersed

among the all daughter

strands

Bacteriacultured inmedium with15N (heavyisotope)

Bacteriatransferred tomedium with14N (lighterisotope)

DNA samplecentrifugedafter firstreplication

DNA samplecentrifugedafter secondreplication

Less dense

More dense

Predictions: First replication Second replication

Conservativemodel

Semiconservativemodel

Dispersivemodel

21

3 4

EXPERIMENT

RESULTS

CONCLUSION

Testing the

ModelsIn this experiment,

bacteria with DNA

containing the “heavy”

isotope 15N were

allowed to reproduce

in medium containing

lighter 14N.

Density-gradient

centrifugation revealed

that DNA replication is

semiconservative.

Matthew Meselson &

Franklin Stahl, 1958

(a) Origin of replication in an E. coli cell

Origin ofreplication Parental (template) strand

Double-strandedDNA molecule

Daughter (new) strand

Replication forkReplicationbubble

Two daughterDNA molecules

0.5 m

DNA Replication in Bacteria

Initiation of DNA

replication

requires an origin

of replication

(b) Origins of replication in a eukaryotic cell

Origin of replicationDouble-strandedDNA molecule

Parental (template)strand

Daughter (new)strand

Bubble Replication fork

Two daughter DNA molecules0.25 m

DNA Replication in Eukaryotes

Eukaryotic DNA

replication

requires multiple

origins of

replication

Leadingstrand

Laggingstrand

Overview

Origin of replicationLaggingstrand

Leadingstrand

Primer

Overall directionsof replication

Overview of DNA Replication

New strand Template strand

Sugar

Phosphate Base

Nucleosidetriphosphate

DNApolymerase

Pyrophosphate

5

5

5

5

3

3

3

3

OH

OHP P i

2 P i

A

A

A

A

T T

T

C

C

C

C

C

C

G

G

G

G

DNA Replication proceeds 5’ to 3’

DNA

polymerase

can add

only to the

3’ end

Topoisomerase

Primase

RNAprimer

Helicase

Single-strand bindingproteins

5

3

5

53

3

Enzymes involved in DNA Replication

DNA Polymerase – synthesizes new DNA

Helicase – unwinds DNA double helix

Topoisomerase – relieves tension due to DNA

unwinding

Primase –

makes short

RNA primers

DNA Ligase –

connects DNA

fragments

Origin of replication

RNA primer

Sliding clamp

DNA pol IIIParental DNA

3

5

5

33

5

3

5

3

5

3

5

Leading Strand DNA SynthesisProceeds toward unwinding replication fork:

DNA polymerase

can only

synthesize DNA

in a 5’ to 3’

direction.

DNA polymerase

requires an RNA

primer which it

can extend in a

continuous

manner toward

the unwinding

replication fork

DNA synthesis is

continuous on

the leading

strand only

Templatestrand

RNA primerfor fragment 1

Okazakifragment 1

RNA primerfor fragment 2

Okazakifragment 2

Overall direction of replication

3

3

3

3

3

3

3

3

3

3

3

3

5

5

5

5

5

55

55

55

5

2

2

21

1

1

1

1

Lagging Strand DNA SynthesisProceeds away from the

unwinding replication fork:

• DNA polymerase

synthesizes DNA in

Okazaki fragments

• each fragment requires

an RNA primer

• another DNA

polymerase will replace

the RNA with DNA

• DNA ligase will link the

fragments together

DNA synthesis is

discontinuous

on the lagging

strand

Overview

Leadingstrand

Origin of replication Lagging

strand

LeadingstrandLagging

strand Overall directionsof replicationLeading strand

DNA pol III

DNA pol III Lagging strand

DNA pol I DNA ligase

PrimerPrimase

ParentalDNA

5

5

5

5

5

3

3

3

333 2 1

4

Summary of DNA Replication

DNA replication proceeds in this manner in ALL

living organisms.

Parental DNA

DNA pol III

Leading strand

Connectingprotein Helicase

Lagging strandDNA pol III

Laggingstrandtemplate

5

5

5

5

5

5

3 3

33

3

3

Current Model of DNA Replication

Nuclease

DNA polymerase

DNA ligase

5

5

5

5

5

5

5

5

3

3

3

3

3

3

3

3

DNA Repair

When DNA is damaged it is

essential that the DNA is

repaired so it can be replicated

and expressed properly.

• special enzymes recognize and

remove the damaged portion of DNA

• a DNA polymerase will fill in the gap

• DNA ligase will then connect the

newly made DNA to the adjacent

strand

Ends of parentalDNA strands

Leading strand

Lagging strand

Last fragment Next-to-last fragment

Lagging strand RNA primer

Parental strandRemoval of primers andreplacement with DNAwhere a 3 end is available

Second roundof replication

Further roundsof replication

New leading strand

New lagging strand

Shorter and shorter daughter molecules

3

3

3

3

3

5

5

5

5

5

The Problem with TelomeresThe ends of linear

chromosomes, the

telomeres, cannot be

completely copied on

the lagging strand.

This results in

progressive shortening

of the chromosome

every time it is

replicated.

Telomerase will solve

this problem in certain

cell types

…more on Chromatin

Chromatin refers to the complex of DNA and

histone proteins in eukaryotic nuclei:

• chromosomal DNA wraps around histone

proteins to form structures called nucleosomes

that look like “beads on a string”

• different parts of a chromosome can be in

various states of “packing”

EUCHROMATIN – loosely packed DNA

HETEROCHROMATIN – tightly packed DNA

Key Terms for Chapter 16

• conservative, semiconservative, dispersive

• bacterial transformation, bacteriophage

• double helix, anti-parallel, sugar-phosphate

backbone

• DNA replication, template

• DNA polymerase, helicase, topoisomerase,

primase, DNA ligase

• pyrimidine, purine, base-pair, complementary

• x-ray crystallography

• leading, lagging strand; continuous, discontinuous

• Okazaki fragment, telomere, telomerase

• chromatin, nucleosome, euchromatin, heterochromatin

Relevant

Chapter

Questions 1-7, 9