BIO 110 Part 14: DNA Replication

Duplication of Genetic Material

 Basic functions of genetic material requires
accurate replication
 Watson and Crick noticed something in the
structure:
 “It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material.”
 Uses principle of complementarity
Figure 14.11
Meselson Stahl experiment
Meselson-Stahl movie
 Semiconservative
 Semiconservative replication: each strand
in a double helix serves as a template
Produces a new complementary strand
Conserves one “old” strand in new duplexes
 Uses the principle of complementary
base pairing: depends on H-bonding
patterns of bases
5’ATGCCGTTACGAATTCTGCTA 3’
3’TACGGCAATGCTTAAGACGAT 5’
 Basic Mechanism
 Open up helix
 Copy each strand
 produces 2 new strands that are each hybrids
of one new and one old strand
 Requirements for Replication
 What do we need to replicate DNA???

1. Template: DNA to copy

2. Monomers: nucleotides
3. Enzymes: DNA polymerase, DNA primase
4. Open helix (break H bonds): helicase
5. Wind / unwind: topoisomerase

 More as we see how process works

 DNA Polymerases
 Many DNA polymerases exist
5 in bacteria
15 in Eukaryotes
 most eukaryotic polymerases are involved
in DNA repair, not replication
 Polymerase III is the main polymerase that
does DNA replication in all organisms
 DNA Polymerase
 Functions:
 copy or repair DNA
 All DNA polymerases:
 synthesize DNA only in 5’ to 3’ direction
Add new nucleotides to the 3’ end
Cannot initiate replication on a single
stranded template
Require primer: short preexisting double
stranded sequence of template
 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
New New
Template strand strand Template strand strand
Incorporation of a nucleotide

HO 3’ 5’ HO 3’ 5’
C G P C G P
O O
O O
P P
T A P T A P
O O
O DNA O
P P
A T polymerase T
P III A P
O O
O
P O P
C G C G
P P
O O
O O
P P
A 3’ OH A T PP
P
O
P O T P
O
A O P PP A 3’ OH
O OH O
P P
5’ 5’
Figure 14.13
 Priming
 RNA Polymerases can initiate synthesis
without a primer... so priming for DNA
synthesis uses an RNA polymerase
 Main enzyme used during replication:
Primase: a replication associated RNA pol
 Consequence: newly synthesized DNA
has RNA at the start...
 must remove the RNA & replace with DNA
Use 5’ 3’ exonuclease of DNA Pol I
Priming of DNA Synthesis
 Implications of Polarity and
Polymerases
 DNA has two antiparallel strands and...
 DNA polymerases only synthesize 5’ to 3’
Synthesize one strand (3’ to 5’ template)
continuously: leading strand
Other strand (5’ to 3’ template) must be
discontiouous, in fragments: lagging
 Need periodic priming on lagging strand
 Call short fragments: Okazaki fragments
Semidiscontinuous synthesis

)
Activities on the Lagging Strand
 Make in pieces and stitch
together
 Need continual primase activity
for lagging strand: makes
primers for each fragment
 Need DNA Pol I:
To remove primers and
replace with DNA
 Need DNA ligase:
 to join fragments
Lagging strand activities

Fig.
 The Replication Fork
 The Replication Fork:
The area where the helix is opening
The place where synthesis is active
 The cell has an organelle for DNA
synthesis:
Contains all the necessary components for
replication of DNA
 Polymerase and other proteins
 accessory proteins
 arranged for coordinated activities
 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Replication Fork Fig.14.19

(new) Figure 14.19

3’

5’

3’
5’

5’
3’
 Other Activities of DNA Pol’s
 Some DNA polymerases also have
nuclease activity
 Nucleases cleave phosphodiester bonds
 Polymerase have exonucleases:
 “chew” away at free end
 this is the 5’ to 3’ or 3’ to 5’ exo
 Both remove bases from only one strand
of double helix
 3’-5’ Exonucleases

 3’5’ exonuclease
 has proofreading function
 if wrong base is inserted by polymerase,
can’t base pair with other strand
This exonuclease removes “wrong base”
DNA pol III (& others)
Proofreading by 3’5’ Exonuclease
 5’-3’ exonucleases
 5’3’ exonuclease
 Removes RNA primer
segments replaces with
DNA nucleotides
 removes damaged
nucleotides
 DNA Pol I & others
 Accuracy of Replication
 Replication is quite accurate, but consider
how much DNA we have...
 Even 1 error in 106 bases would be bad
(we have 3 x 109 bp of DNA!)
 error checking and repair can lower error
frequencies to 10^8, 10^9

 Checking replication accuracy
 What are the checks on accuracy of
base pairing:
1. selecting correct base
2. removal of incorrectly paired nucleotide by
the proofreading activity
3. Repair of mismatched base pairs by a
post-replication DNA repair mechanism
 Consequences of Errors
 Replication errors = mutations
 This explains the source of the large
amount of variation we see
 mutations are constantly arising due to
replication errors
 can also arise by misrepair of damaged
DNA
 Not what individual animals want...
 Provides variation acted on by evolution
 Point Mutations
 Point mutations: two types
1. single base & substitution
2. Deletion or insertion of single/couple bases
 Base substitutions:
Transitions: purine to purine or pyrimidine to
pyrmidine e.g. A <—> G or C <—> T
Transversions: purine to pyrimidine or
pyrimidine to purine e.g. C,T <—> A,G
thebigfatcatatetherat
thebigfatcatatethebat
thebigfztcatatetherat
 Mutational indels
 Small insertions/deletions:
 can cause frame shifts, changes reading
frame of message
The big fat cat ate the rat
The bif atc ata tet her at

 whole segments of a gene product may be

missing
 Additional amino acids may be introduced
into the gene product
 Larger Genetic Changes
Chromosomal Rearrangements:
Large deletions: loss of DNA
Inversions: reversal of gene order
Duplications: duplication of existing
DNA
 Consequences of these chromosomal
rearrangements are hard to predict
May depend on where chromosomes are
“broken” & rearranged
 Chromosome Rearrangements
 Original Chromosome

Deletion

Inversion

Duplication
 DNA mutagens
 DNA experiences mutations from
occasional replication errors
 Mutagens: agents that cause a higher
than normal rate of mutations in DNA
Carcinogens: mutagens that cause cancer
 Mutagenic affects:
Different patterns of DNA mutation for
different mutagens
 Mutations and Humans
 Mutations produce new alleles, but most
are deleterious
 What do mutations mean in terms of
human health?
Germ line mutations = heritable genetic
diseases
Somatic mutations = cancerous tumor
growth (tumorigenesis)
 Diseases can be due to single-gene
mutations
 Heritable Genetic Diseases
 Examples:
 Cystic fibrosis: chloride channel function
Tay Sachs: failure of lipid metabolism--
accumulate abnormal brain lipids (lethal)
Hemophilia: X-linked recessive; clotting
factor VIII
Xeroderma pigmentosum-mutation in
mechanisms for repairing UV damage to
DNA—leads to melanomas
Plus 1,000s more
 Gene x environment interactions
 Genetic diseases:
Complex relationship between genotype and
phenotype (disease state)
May be caused by the collective effects of
multiple genes
Only manifested in a particular environment

Diet, exercise, lifestyle a key contributor to

many diseases
 Cancer
 Cancer is a multi-step process
 Not completely understood, but have
working models
 Cell division cycle is under genetic control
 Cancer is caused by disruption, or errors
in this control system
 Affected by both genetics and
environment
 “Cancer Genes”
 Proto-Oncogenes: normal signals to stimulate
cell growth and division
 e.g. growth factors & GF receptors
 Mutated oncogenes overproduce stimulatory
products
Lead to tumor growth
 Tumor Suppressor gene: signals slow cell
growth and division rates
 May be linked to damage sensing
 Mutations fail to balance normal stimulatory
signals; loss leads to tumor growth
Cell growth accelerators and inhibitors

Oncogenes
stimulate cell
growth &
division

Tumor
supressor
genes slow
growth & cell
division