Notes

Week 2 – DNA replication
and recombination
BIOL2301: Genetics
Teaching team: Sara Good
May 17th, 2024
Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Agenda today
1. Overview of Genomes and their diversity
2. Mechanism of replication based on DNA structure.
3. Different modes of replication, replication requirements,
and directions of synthesis.
4. Process of replication

Are genomes structured the same?
what is a genome?
a set of genetic instructions

within a biological compartment
How many distinct genomes
can a eukaryote have?
?
What genetic compartments?
Archaea
Bacteria
nuclei
chloroplasts
viruses
in eukaryotes…
nuclear
genome
1 nucleus
DNA
DNA
2
mitochondrial mitochondrion
genome
in eukaryotes…
nuclear
genome
1 nucleus
DNA
DNA
DNA
2 chloroplast
mitochondrion
mitochondrial
genome
3 15
?
chloroplast
genome
in eukaryotes…
nuclear
genome
1 nucleus
DNA Polytomella
DNA
2 chloroplast
mitochondrion
mitochondrial
genome chloroplast
genome loss Rafflesia
Why keep organelles that have
lost their genome?
mitochondrion chloroplast
Presumably still perform some crucial functions

Endosymbiosis
symbiont
chloroplast
nucleus nucleus
GENETIC MERGER
1) Feeding
2) Endosymbiosis
3) Sharing
4) Entrapment
5) Transfer of control
6) Genetic Integration … thousands of years go by
Recall….
symbiont
partial
nucleus
chloroplast
nucleus
GENETIC MERGER
1) Feeding
2) Endosymbiosis
3) Sharing
4) Entrapment
5) Transfer of control
6) Genetic Integration … still in progress – integration not complete!
in eukaryotes…
nucleomorph
nuclear genome
genome
1 nucleus 4
DNA
nucleomorph Cryptomonad
DNA
DNA
2
mitochondrion 2° chloroplast
mitochondrial
genome
Chlorarachiophyte
3
secondary
chloroplast
genome
# of genomes within
a single organism
can vary!
How do genomes vary in
structure?
?
* not to scale
RNA DNA
circular linear
genomes can be:
INTACT FRAGMENTED
How would you define A) the human nuclear

? genome and B) the human mitochondrial genome?
genomes can be:
MIXTURE OF LINEAR &

CIRCULAR
(example: Borrelia)
Chromosomes can be:
LINKED CIRCLES
(CHAINMAIL)
(example: Trypanosome)
Chromosomes can be:
too complex to define
highly branched structure

Linear or circular
… why does it matter?
easy to replicate
hard to replicate
telomeres are prone to damage

origin of replication
termination of
replication
easy to replicate
termination of
replication
5’ 3’
3’ 5’
This is called
the end replication problem
shortening of ends
TELOMERES
5’ 3’
3’ 5’
5’ 3’
3’ 5’
shortening of ends
TELOMERES
How do we
Telomerase
do it?
Are there other solutions?
Borrelia
Telomeres Telomeres
5´ 3´
3´ 5´
Chromosome ends are closed single-stranded loops.

Helps overcome end replication problem and protects telomeres.
So why have linear at all?
Chromosomes don’t really
look like this
E. coli
nuclear
chromosomes
DNA is rarely “naked”
inside of cells
It is typically tightly packed
with proteins
With linear, this

packing produces
less torque
Or linearization could happen by
chance, then unable to be undone
evolutionary ratchet
genomes can be:
INTACT FRAGMENTED
How much does this vary?

chromosome # varies a lot!
46
one chromosome many chromosomes
Jack jumper ant 254: hermit crabs
1260: Ophioglossum fern

How can we get variation in
chromosome number without ploidy
changes?
Robertsonian
translocation
Would have to fusion
split centromere
fission
same
genetic
info
Chromosome # can even vary within species
Blarina carolinensis shrew 21 shrews tested

n=
35
36 Robertsonian
37 polymorphisms
38
39
40
41
44 chromosomes
How ok?
fusion of chr. 14 + 15
Chromosome # can even vary within an individual
Normally 2n = 54 Some cells 2n = 54

2n = 55
2n = 56
Trichomycterus davisi fish

How do genomes vary in
size?
what is a genome?
a set of genetic instructions

within a biological compartment
Genome size:
is the length of those instructions

what is genome size?
genetic compartments
genome size
Units of genome size
nt bp
nucleotide vs base pair
single stranded double stranded

Units of genome size
1 base pair (bp) A–T

1,000 base pairs (kb)
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1,000,000 base pairs (Mb)
1,000,000,000 base pairs (Gb)

who has the biggest genome?
complexity
viruses bacteria unicellular multicellular

eukaryotes eukaryotes
tiny small medium big
genome size
complexity
viruses bacteria unicellular multicellular

eukaryotes eukaryotes
tiny small medium big
genome size
X
complexity
eukaryotes
small medium massive
genome size
20 million bp
Nematode:
Pratylenchus coffeae
60 million bp
Carnivorous plant: Genlisea margaretae

150 billion bp
Paris japonica “canopy plant”

130 billion bp
Marbled lungfish
670 billion bp
Amoeba: Polychaos dubium

3 billion bp
Genome size
Microsporidia
smallest nuclear genome
(2 Mb)
eukaryotic microbes
Bacteria
Archaea
Mitochondria
Chloroplasts variation here, too
Viruses
103
-3 -2 4
10 105 106 107 108 109 1010 1011 1012
small Genome size (bp) large
Genome size is:
Hugely variable within

and among lineages.
chloroplasts
This is true for nuclei
all types of genome.

discordance between complexity &
genome size
C-value paradox
What do big genomes have that
little genomes don’t have?
?
sequence some genomes & find out
whole-genome sequencing
3 Gb
2001
100
100 Mb
1998
12 Mb
80 1996
% non-coding
2 Mb
156 kb 1995
60 16 kb 1986
1981
chloroplast
5 kb
1977
40
20
tiny massive
Genome Size
whole-genome sequencing
100
80
% non-coding
60
40
20
tiny massive
Genome Size
What do big genomes have that
little genomes don’t have?
The answer to the C-value paradox
non-coding DNA
* DNA that does not encode
proteins or functional RNAs
intergenic DNA
intronic DNA
43 billion bp
99.9% non-coding
Differences in
gene to bp
ratio
Gene distribution also varies by chromosome
within an organism, called gene density
Human Chr 19 has ~25 genes per million bp.
Human Y Chr has ~1 gene per Mbp
One region on human chromosome 21

has 7 million bp with no genes
Why do some genomes or genome regions have an
abundance of non-coding DNA while others do not?
mystery
replaced C-value paradox
Non-coding has a function
sometimes yes
…but often no
“Selfish” DNA hypothesis
mobile mobile mobile mobile mobile mobile mobile mobile mobile mobile mobile
element element element element element element element element element element element
genome
an evolutionary ratchet?
Mobile element = transposable element

Why not have a big genome?
“Race to replication” hypothesis

selective premium on high replication rates
Metabolic costs of DNA

What do little genomes do to
be small?
?
shorter genes
fewer introns
shorter introns
shorter intergenic spacers
fewer transposable elements
vs.
vs.
fewer genes
overlapping reading frames

- same direction, staggered
- different directions
fewer genes
overlapping reading frames
- nested or embedded
- same or different directions
nested embedded
Mechanism of replication
based on DNA structure
Importance of DNA replication
DNA replication ensures each new cell receives a complete
set of genetic information.

Importance of DNA replication
The precise copying of
eukaryotic DNA is
accomplished by separating the
initiation of replication into two
steps.
STEP 2
Initiation in the S
stage STEP 1
Origin licensing in
the G1 stage
Meier-Gorlin syndrome

Small,
underdeveloped
Meier-Gorlin syndrome and rotated
external ears
High-arched
palate Missing
kneecaps
High
forehead
Small,
Small, underdeveloped
underdeveloped and rotated
ears High forehead external ears
Small,
underdeveloped
Meier-Gorlin syndrome and rotated
external ears

Missing
kneecaps

High
forehead

Small,
underdeveloped
ears
High forehead
Small,
underdeveloped
and rotated
external ears
Watson and Crick’s discovery (1953)

Semiconservative replication
● Semiconservative replication: the process by
which DNA is duplicated, producing two copies each
containing one original strand and one newly
synthesized strand.
○ Each original nucleotide strand remains intact.
○ Original DNA is half conserved in new DNA molecules.

Conservative replication
Entire double-stranded DNA
serves as a template for a new
molecule. Original DNA is fully
conserved.

Dispersive replication
DNA strands break into
fragments and reassemble.
Produces hybrid molecules
with interspersed old and new
DNA.

Semiconservative replication
Intermediate between conservative and
dispersive. Strands unwind and serve as
templates for new DNA. Produces two hybrid
molecules after one round of replication.

Meselson and Stahl’s experiment
● Objective: determine the correct DNA replication
model (conservative, semiconservative, or
dispersive) in E. coli cells.
● Methodology:
○ Utilized two nitrogen isotopes: N¹⁴ (common) and N¹⁵
(rare, heavy).
○ Grew E. coli in N¹⁵ medium, incorporating N¹⁵ into their
DNA.
○ Switched E. coli to N¹⁴ medium and sampled over
generations.
● Technique:
○ Equilibrium density gradient centrifugation used to
distinguish heavy N¹⁵-laden DNA from light N¹⁴-
containing DNA.
○ Centrifuge creates a density gradient: heavy DNA
sinks, light DNA rises.


Different modes of
replication, replication
requirements, and directions
of synthesis.
Modes of replication
● Semiconservative replication confirmed. No evidence
for conservative or dispersive replication.
● Type of template DNA:
○ Linear DNA: typically found in eukaryotic chromosomes.
○ Circular DNA: common in bacterial chromosomes and
some viral genomes.
● Replicon: a DNA segment that undergoes replication.
○ Each replicon has an origin of replication where
replication initiates.
● Replication origins:
○ Bacterial Chromosomes: Single origin of replication.
○ Eukaryotic Chromosomes: Multiple origins of
replication, allowing efficient duplication of large
genomes.
Theta replication
● Theta replication: a common mode of replication that
takes place in circular DNA, such as that found in E.coli
and other bacteria.
○ Named for its resemblance to the Greek letter theta (θ).

Theta replication

Rolling-circle replication
● Rolling-circle replication: used by some viruses and
the F factor in E. coli. Involved in the transfer of genetic
material during bacterial conjugation.

Linear eukaryotic replication
● Linear chromosomes in
eukaryotic cells are too
large to be replicated
from a single origin.
● Linear eukaryotic
replication: the process
by which eukaryotic cells
replicate their linear
chromosomes using
multiple origins of
replication.

Requirements of replication
Template:
single-
stranded
DNA
Major group of
components
included in the
process of
replication
Raw
Enzymes
materials
and
(substrates):
proteins
dNTPs

Raw materials:
dNTPs (consist of
a deoxyribose
sugar, a base, and
three phosphate
groups)

Nucleotides are
added to the 3′-OH
group of the
growing strand.

Direction of replication
New nucleotides are added to the 3′ end of the growing
DNA strand. DNA polymerases can only add nucleotides to
the 3′ end, not the 5′ end.

● Leading strand:
○ Template direction: Exposed in the 3′ → 5′ direction.
○ Synthesis: Continuous in the 5′ → 3′ direction.
○ Replication: New strand synthesized continuously as the
DNA unwinds.
● Lagging strand:
○ Template direction: Exposed in the 5′ → 3′ direction.
○ Synthesis: Discontinuous in the 5′ → 3′ direction.
○ Replication: Short segments synthesized opposite to the
direction of unwinding. Known as Okazaki fragments.
Synthesis starts anew as more template is exposed.



Process of replication
Initiation Known as DnaA in E.
coli are molecules
that recognize and
bind to the origin of
replication to unwind
and start the DNA
replication process.

Initiation
Short section of DNA

to unwind

Initiation
This unwinding allows

helicase and other
single-strand-
binding proteins to
attach to the
polynucleotide strand

Unwinding - DNA helicase
DNA helicase breaks the hydrogen bonds that exist

between the bases of the two nucleotide strands of a
DNA molecule. Copyright © 2024 Summations Knowledge Inc. All Rights Reserved
Unwinding - single strand binding
proteins
Single-strand-binding proteins stabilize the unwound DNA

strands during replication, preventing them from reannealing or
forming secondary structures.
Unwinding - DNA gyrase
Protein essential for the unwinding process is the enzyme

DNA gyrase, a topoisomerase.
Elongation - the synthesis of
primers
Primase
synthesizes
short RNA
primers
providing the 3′-OH
group for DNA
polymerase.

primers
A single RNA
primer is
needed at the
5′ end for continuous synthesis
on the leading strand.

Multiple RNA primers
primers are needed for each
Okazaki fragment on
the lagging strand.

Elongation - DNA synthesis by DNA
polymerases
● DNA polymerases role: DNA polymerases elongate new
DNA strands after unwinding and primer addition.
● E. coli DNA Polymerases: Five types, with DNA
polymerase I and III involved in replication, others in repair.
● DNA polymerase III:
○ Main Workhorse: synthesizes DNA by adding nucleotides to
the 3′ end.
○ Activities:
■ 5′ → 3′ Polymerase: Adds new nucleotides.
■ 3′ → 5′ Exonuclease: Corrects errors by removing incorrect
nucleotides.
○ High Processivity: Can add many nucleotides without releasing
the template.
○ β Sliding Clamp: Ensures high processivity by allowing easy
sliding along the template.
○ DNA replication
polymerases
● DNA polymerase I:
○ Activities:
■ 5′ → 3′ Polymerase and 3′ → 5′ Exonuclease: Synthesize
DNA and correct errors.
■ 5′ → 3′ Exonuclease: Removes RNA primers and replaces
them with DNA.
○ Lower Processivity: Mainly removes and replaces
primers.

polymerases

Elongation - primer
replacement and DNA
ligase
Continuous Addition of DNA Nucleotides

DNA polymerase III attaches DNA
nucleotides to the 3′-OH group of the RNA
primer, continuing as long as the template
is available.

Elongation - primer
replacement and DNA
ligase
RNA Primer Removal and Replacement

DNA polymerase I uses its 5′ → 3′
exonuclease activity to remove RNA
primers and its 5′ → 3′ polymerase
activity to replace them with DNA
nucleotides.

Elongation - primer
replacement and DNA
ligase
Formation of a Nick
After the RNA primer is
replaced, a nick remains in the
sugar-phosphate backbone
due to the unattached 3′-OH
group of the last DNA
nucleotide added by DNA
polymerase I.

Elongation - primer
replacement and DNA
ligase
Sealing the Nick with DNA Ligase
DNA ligase seals the nick by catalyzing a
phosphodiester bond, completing the DNA
strand without adding a new nucleotide.

Termination
● Termination: replication
is terminated when two
replication forks meet.
● Ter Sites: Specific
termination sequences
that block further
replication.
● Tus-Ter Complex: In E.
coli, Tus protein binds to
Ter sites to form a
complex that halts
helicase movement.

Notes

Uploaded by

Document Informationclick to expand document informationclass notes

Document Informationclick to expand document information

Copyright:

Available Formats

Notes

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Notes

Uploaded by

Copyright:

Available Formats

Week 2 – DNA replication

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

a set of genetic instructions

Presumably still perform some crucial functions

How would you define A) the human nuclear

MIXTURE OF LINEAR &

too complex to define

highly branched structure

telomeres are prone to damage

Chromosome ends are closed single-stranded loops.

With linear, this

How much does this vary?

Jack jumper ant 254: hermit crabs

1260: Ophioglossum fern

Blarina carolinensis shrew 21 shrews tested

Normally 2n = 54 Some cells 2n = 54

Trichomycterus davisi fish

a set of genetic instructions

is the length of those instructions

single stranded double stranded

1 base pair (bp) A–T

1,000,000 base pairs (Mb)

1,000,000,000 base pairs (Gb)

viruses bacteria unicellular multicellular

tiny small medium big

viruses bacteria unicellular multicellular

tiny small medium big

small medium massive

Carnivorous plant: Genlisea margaretae

Paris japonica “canopy plant”

Amoeba: Polychaos dubium

Hugely variable within

all types of genome.

The answer to the C-value paradox

Human Chr 19 has ~25 genes per million bp.

Human Y Chr has ~1 gene per Mbp

One region on human chromosome 21

Mobile element = transposable element

“Race to replication” hypothesis

Metabolic costs of DNA

shorter intergenic spacers

fewer transposable elements

overlapping reading frames

overlapping reading frames

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved

Copyright © 2024 Summations Knowledge Inc. All Rights Reserved