Principles of Biology

SCPBA1

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Week 5: DNA replication
DNA
Life’s operating instructions – self study
• In 1953, James Watson and Francis Crick introduced an elegant double-
helical model for the structure of deoxyribonucleic acid, or DNA
• Hereditary information is encoded in DNA and reproduced in all cells of the
body
• This DNA program directs the development of biochemical, anatomical,
physiological, and
(to some extent) behavioral traits
Search for genetic material – self study
• When T. H. Morgan’s group showed that genes are located on
chromosomes, the two components of chromosomes—DNA and protein—
became candidates for the genetic material

• The role of DNA in heredity was first discovered

by studying bacteria and the viruses that
infect them
Evidence that DNA can transform
Bacteria – self study
• The discovery of the genetic role of DNA began with research by Frederick
Griffith in 1928
• Griffith worked with two strains of a bacterium, one pathogenic and one
harmless
• When he mixed heat-killed remains of the pathogenic strain with living cells
of the harmless strain, some living cells became pathogenic
• He called this phenomenon transformation, now defined as a change in
genotype and phenotype due to assimilation of foreign DNA
Evidence that viral DNA can program cells – self study
• More evidence for DNA as the genetic material came from studies of
viruses that infect bacteria
• Such viruses, called bacteriophages (or phages), are widely used in
molecular genetics research
• A virus is DNA (sometimes RNA) enclosed by a protective coat, often simply
protein
• In 1952, Alfred Hershey and Martha Chase showed that DNA is the genetic
material of a phage known as T2
• They designed an experiment showing that only one of the two
components of T2 (DNA or protein) enters an E. coli cell during infection
• They concluded that the injected DNA of the phage provides the genetic
information
Building the structure of DNA – self study
• After DNA was accepted as the genetic material, the challenge was to
determine how its structure accounts for its role in heredity
• Maurice Wilkins and Rosalind Franklin were using a technique called X-ray
crystallography to study molecular structure
• Franklin produced a picture of the DNA molecule using this technique
• Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce
that DNA was helical
• The X-ray images also enabled Watson to deduce the width of the helix and
the spacing of the nitrogenous bases
• The pattern in the photo suggested that the DNA molecule was made up of
two strands, forming a double helix
DNA as a genetic material
Figure 16.7

5′ end
C G
C G Hydrogen bond 3′ end
G C
G C T A

3.4 nm
T A
G C G C
C G
A T

1 nm C G
T A
C G
G C
C G A T

A T 3′ end
A T
T A
0.34 nm 5′ end
(a) Key features of (b) Partial chemical structure (c) Space-filling
DNA structure model
DNA – Chargaff’s rule
• Two findings became known as Chargaff’s rules
✓ The base composition of DNA varies between species
✓ In any species the number of A and T bases are equal and the number of G
and C bases are equal

• The basis for these rules was not understood until the discovery of the
double helix
DNA – Watson and Crick’s model
• Watson and Crick built models of a double helix to conform to the X-rays
and chemistry of DNA
• Franklin had concluded that there were two outer sugar-phosphate
backbones, with the nitrogenous bases paired in the molecule’s interior
• Watson built a model in which the backbones were antiparallel (their
subunits run in opposite directions)
• At first, Watson and Crick thought the bases paired like with like (A with A,
and so on), but such pairings did not result in a uniform width
• Instead, pairing a purine with a pyrimidine resulted in a uniform width
consistent with the X-ray data
DNA – Watson and Crick’s model
• Watson and Crick reasoned that the pairing was more specific, dictated by
the base structures
• They determined that adenine (A) paired only with thymine (T), and
guanine (G) paired only with cytosine (C)
• The Watson-Crick model explains Chargaff’s rules: in any organism the
amount of A = T, and the amount of G = C
DNA Replication
Base pairing to template strand
• Since the two strands of DNA are complementary, each strand acts as a
template for building a new strand in replication
• In DNA replication, the parent molecule unwinds, and two new daughter
strands are built based on base-pairing rules
Watson and Crick’s model
• Watson and Crick’s semiconservative
model of replication predicts that when
a double helix replicates, each daughter
molecule will have one old strand
(derived or “conserved” from the parent
molecule) and one newly made strand
• Competing models were the
conservative model (the two parent
strands rejoin) and the dispersive model
(each strand is a mix of old and new)
Activity
• Describe the process of DNA replication by drawing a flow diagram
DNA Replication
• The copying of DNA is remarkable in its speed and accuracy
• More than a dozen enzymes and other proteins participate in DNA
replication
• Replication begins at particular sites called origins of replication, where the
two DNA strands are separated, opening up a replication “bubble”
• A eukaryotic chromosome may have hundreds or even thousands of origins
of replication
• Replication proceeds in both directions from each origin, until the entire
molecule is copied
Figure 16.12

(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell
Origin of Parental (template) Origin of
replication strand replication Eukaryotic chromosome
Daughter
(new) strand Parental (template)
Double-stranded strand
Replication DNA molecule Daughter (new)
Bacterial
fork strand
chromosome
Double-
Replication
stranded
bubble Replication
DNA molecule Bubble
fork

Two daughter
DNA molecules

Two daughter DNA molecules

0.25 µm
0.5 µm
DNA Replication
• At the end of each replication bubble is a replication fork, a Y-shaped
region where new DNA strands are elongating
• Helicases are enzymes that untwist the double helix at the replication forks
• Single-strand binding proteins bind to and stabilize single-stranded DNA
• Topoisomerase corrects “overwinding” ahead of replication forks by
breaking, swiveling, and rejoining DNA strands
DNA Replication
• Enzymes called DNA polymerases catalyze the elongation of new DNA at a
replication fork
• Most DNA polymerases require a primer and a DNA template strand
• The rate of elongation is about 500 nucleotides per second in bacteria and
50 per second in human cells
• DNA polymerases cannot initiate synthesis of a polynucleotide; they can
only add nucleotides to an existing 3′ end
• The initial nucleotide strand is a short RNA primer
• An enzyme called primase can start an RNA chain from scratch and adds
RNA nucleotides one at a time using the parental DNA as a template
• The primer is short (5–10 nucleotides long), and the 3′ end serves as the
starting point for the new DNA strand
DNA Replication
• Each nucleotide that is added to a growing DNA strand is a nucleoside
triphosphate
• dATP supplies adenine to DNA and is similar to the ATP of energy
metabolism
• The difference is in their sugars: dATP has deoxyribose while ATP has ribose
• As each monomer of dATP joins the DNA strand, it loses two phosphate
groups as a molecule of pyrophosphate
DNA Replication
• The antiparallel structure of the double helix affects replication
• DNA polymerases add nucleotides only to the free 3′ end of a growing
strand; therefore, a new DNA strand can elongate only in the 5′ to 3′
direction
• Along one template strand of DNA, the DNA polymerase synthesizes a
leading strand continuously, moving toward the replication fork
• To elongate the other new strand, called the lagging strand, DNA
polymerase must work in the direction away from the replication fork
• The lagging strand is synthesized as a series of segments called Okazaki
fragments, which are joined together by DNA ligase
DNA Replication
• The proteins that participate in DNA replication form a large complex, a
“DNA replication machine”
• The DNA replication machine may be stationary during the replication
process
• Recent studies support a model in which DNA polymerase molecules “reel
in” parental DNA and “extrude” newly made daughter DNA molecules
Proofreading and repairing DNA
• DNA polymerases proofread newly made DNA, replacing any incorrect
nucleotides
• In mismatch repair of DNA, repair enzymes correct errors in base pairing
• DNA can be damaged by exposure to harmful chemical or physical agents
such as cigarette smoke and X-rays; it can also undergo spontaneous
changes
• In nucleotide excision repair, a nuclease cuts out and replaces damaged
stretches of DNA
Evolutionary significance of altered
DNA nucleotides
• Error rate after proofreading repair is low but
not zero
• Sequence changes may become permanent and can be passed on to the
next generation
• These changes (mutations) are the source of the genetic variation upon
which natural selection operates
Reminders