CHAPTER 6

RECOMBINANT/
DNA REPAIR

At the end of the lecture, students should be able to:

Explain the mechanism of DNA replication
Describe the DNA replication

Holliday Model for Homologous Recombination

DNA damage and repair

Alkylation of bases
Ultraviolet damage
Gamma and X-rays
Excision repair (Base and nucleotide)
Mismatch repair (Wrong base entered)

Three Hypotheses of Replication

The three methods of
DNA replication
considered were:
1. Semiconservative
2. Conservative
3. Dispersive

20-3

Meselson-Stahl experiment
Meselson and Stahl carried out experiment to

prove that DNA replication is

semiconservative.
To discriminate these three models, they had to
distinguish between old and newly synthesized
DNA
For the first step, they used heavy isotope of
nitrogen 15N to differentiate these strands

Meselson-Stahl experiment
The 15N isotope contains an extra neutron on

its isotope giving it a higher atomic weight

than the more abundant light isotope 14N.
Nitrogen atoms made up the purine and
pyrimidine bases in DNA, E. coli DNA
(experiment organism) was grown in a
medium containing 15N ammonium salts
as the sole nitrogen source.
After several generations of growth, the
bacteria contained only 15N labeled DNA.

Meselson-Stahl experiment
After several generations of growth in a

medium containing 15N labeled DNA, the E.

coli were transferred to a medium
containing the normal light 14N isotope
Samples were removed from the cultures
periodically and analyzed by equilibrium
density-gradient centrifugation in CsCl to
separate heavy-heavy (H-H), light-light (L-L)
and heavy-light (H-L) duplexes into distinct
bands

Meselson-Stahl experiment

Meselson-Stahl experiment

Semiconservative Replication
DNA replicates in a
semiconservative manner
When parental strands
separate
Each strand serves as
template
Makes a new, complementary
strand

DNA replication
DNA replication is the process of copying a

double-stranded DNA molecule.

In a cell, DNA replication must happen before
cell division.
Prokaryotes replicate their DNA throughout the
interval between cell divisions.
In eukaryotes, timings are highly regulated and
this occurs during the S phase of the cell cycle,
preceding mitosis or meiosis I.

DNA replication
DNA replication begins with a partial unwinding of

the double helix at an area known as the

replication fork.
This unwinding is accomplished by an enzyme
known as DNA helicase
The two parental DNA will separate forming
replication bubbles
Replication of DNA proceeds in both direction
until both DNA molecule is copied
Multiple replication bubbles will fuse speeding up
the process of copying long DNA strand

DNA replication
As the two DNA strands separate

("unzip") and the bases are exposed, the

enzyme DNA polymerase moves into
position at the point where synthesis
will begin.
Single strand binding protein will
bind to the single stranded DNA and
stabilize it

DNA replication
The start point for DNA polymerase is a short

segment of RNA known as an RNA primer.

The term primer is indicative of its role which is
to prime or start DNA synthesis at certain
points.
The primer is laid down complementary to the
DNA template by an enzyme known as RNA
polymerase or Primase.

DNA replication
DNA polymerase III then adds nucleotides one

by one in an exactly complementary manner, A to

T and G to C
DNA polymerase will read the sequence of bases
on the template strand and then synthesize the
complementary strand.
The template strand is read in the 3' to 5 direction
The new DNA strand (since it is complementary)
will be synthesized in the 5' to 3' direction
This strand is called the leading strand

DNA replication
As for the other strand, replication (synthesizing)

still have to happen from the 5 to the 3 end

therefore it worked away from the replication fork
Replication is carried out by DNA polymerase III
This strand is called the lagging strand and
replication happens in a series of segment called
Okazaki fragment
DNA Polymerase I removes the primer from
the lagging strand and replace it with DNA
Another enzyme called DNA ligase will joint the
Okazaki fragments (with newly synthesized DNA
fragments)

Homologous Recombination
Occurs between homologous chromosomes during
meiosis
The process scrambles the genes of maternal and
paternal chromosomes resulting in nonparental
combinations in the offspring
Meiotic recombination forms physical links between
homologous chromosomes that allow them to align
properly during meiotic prophase so they separate
properly during meiotic metaphase
It also plays an important role in allowing cells to
deal with DNA damage by recombination repair

Holliday Model for recombination

The mechanism of homologous recombination

was mainly obtained from the study ofE. coli.

Although bacteria do no undergo meiosis,
homologous recombination could occur during
or immediately after DNA replication.
When two homologous DNA molecules line
up /two nonsister chromatids line up during
meiosis.
There are cuts in one strand of both DNAs.

Holliday Model for recombination

The cut strands cross and join homologous strands,

forming the Holliday structure (or Holliday junction).

Resulted in the formation of heteroduplex (A DNA molecule
with two constitutive strands that derived from distinct
sources) by branch migration.
DNA strands may be cut along either the vertical line or
horizontal line.
The vertical cut will result in crossover between f-f' and FF' regions. The heteroduplex region will eventually be corrected
by mismatch repair.
The horizontal cut does not lead to crossover after mismatch
repair. However, it could cause gene conversion.

Homologous Recombination

Double strand break repair by homologus recombination.mp4

DNA Damage and Repair

DNA can be damaged in many different

ways, if left unrepaired this damage

can lead to mutation, changes in the
base sequence of DNA
DNA damage is not the same as
mutation though it can lead to mutation
If a particular kind of DNA damage is
likely to lead to a mutation, we call it
genotoxic

DNA Damage and Repair

DNA damage is chemical alteration (from G-C

to ethyl G-C)
DNA mutation (From G-C to A-T)

Definition of DNA Damage

DNA damage is a chemical alteration
Mutation

is a change in a base pair

Common

examples of DNA damage

Base modifications caused by

alkylating agents
Pyrimidine dimers caused by UV
radiation

Damage Caused by Alkylation of

Bases
Alkylation is a

process where
electrophiles:

Encounter negative
centers (in yellow)
Attack them
Add carbon-containing
groups (alkyl groups)

Damage Caused by Alkylation of Bases

N7 alkylation of guanine did not change the

base-pairing properties
N3 alkylation produced 3-methyl adenine
[3mA] that cannot base pair with any other
bases thus stalling DNA replication (noncoding
base)
Lead to mutation
Cells that lose control over replication may lead
to cancer

Damage Caused by Alkylation of Bases

Alkylating agents like ethylmethane sulfonate (EMS)
add alkyl groups to bases

Some alkylation dont change base-pairing

Others cause DNA replication to stall

Cytotoxic
Lead to mutations if cell attempts to replicate without damage
repair

Third type change base-pairing properties of a base, so

are mutagenic

Damage Caused by Radiation

Ultraviolet rays

Comparatively

low energy
Result in formation of pyrimidine dimers, also
called cyclobutane pyrimidine dimers (CPDs)
Gamma and x-rays
Much more energetic
Ionize molecules around the DNA
Form highly reactive free radicals that attack
DNA
Alter bases
Break strands

DNA Damage:
Pyrimidine Dimers
These lesions
(dimers) block DNA
replication (noncoding)
What base to
insert?
Wrong bases
inserted may lead
to mutations.

DNA Damage:
Pyrimidine Dimers

Damage by Gamma and X-rays

Ionizing the molecules esp water

Form free radicals which are substance without

paired electron
8-oxoguanine will be read as thymine genotoxic
mutation (altering base /breaking strands)

DNA repair
Two basic ways
to do it
Directly
underdoing
it
Remove
damaged
section and
replace a
new one

Directly Undoing UV
DNA Damage (Kelner 1940s)
UV radiation damage to DNA

can be directly repaired by

photoreactivation - a
photolyase, which is
actually two separate
enzymes that catalyze repair
of CPDs
Uses energy from near-UV
to blue light to break bonds
holding 2 pyrimidines
together

Directly Undoing UV
DNA Damage

Two enzymes responsible are CPD photolyase

and (6-4) photolyase

CPD photolyase binds to the lesions, absorb
light in the UV-A to blue region, breaking the
bonds (not in placental mammals)
Humans, another kind of damage by Gamma
and X rays, O6-methyl guanine
methyltransferase by accepting the
methyl/ethyl group (suicide enzyme)

Excision Repair
Percentage of DNA damage products that

can be handled by direct reversal is small

Most damage involves neither pyrimidine
dimers nor O6-alkylguanine
Another repair mechanism is required,
excision repair is the process that
removes most damaged nucleotides

Damaged DNA is removed

Replaced with fresh DNA
Base and nucleotide excision repair are both used,
BER and NER, respectively

Base Excision Repair in E.

coli
DNA glycosylase

removed base from the

sugar apurinic or
apyrimidinic

AP endonuclease cuts

or nicks DNA strand

Phosphodiesterase
removes the AP sugar
phosphate
DNA Polymerase I
performs repair
synthesis
DNA ligase seals the gap

Base Excision Repair

Base excision repair (BER) acts on subtle base
damage
Begins with DNA glycosylase
Extrudes a base in a damaged base pair
Clips out the damaged base
Leaves an apurinic or apyrimidinic site that
attracts DNA repair enzymes
DNA repair enzymes
Remove the remaining deoxyribose phosphate
Replace it with a normal nucleotide

Nucleotide Excision Repair

Nucleotide excision repair typically

handles bulky damage that distorts DNA

double helix (pyrimidine dimers)
NER in E. coli begins when damaged DNA
is clipped by an endonuclease on either
side of the lesion, sites 12-13 nucleotide
apart
Allows damaged DNA to be removed as
part of resulting 12-13-base
oligonucleotide

Nucleotide Excision Repair

NER system

recognizes parental
strand by
methylated A
uvrABC
endonuclease cuts
the bulky damaged
sites
DNA polymerase
and ligase in action

Mismatch repair after replication

(wrong base entered)

Methylated

adenines determines
parental strands
mutH, L and S
introduce nicks.
Exonuclease
removed strand.
DNA pol and ligase.
Methyl transferase
adds the CH3

Coping with DNA Damage Without Repair

Direct reversal and excision repair are

true repair processes

Eliminate defective DNA entirely
Cells can cope with damage by
moving around it
Not true repair mechanism
Better described as damage bypass
mechanism

END OF LECTURE