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DNA REPAIR

PROVIDING CHEMICAL STABILITY FOR LIFE

M. Tofazzal Islam
GEB Department, East West University

DNA A mystery of life


From

one cell to another, from one


generation to the next.
The genetic information that governs how
human beings are shaped has flowed
through our bodies for hundreds of
thousands of years.
It is constantly subjected to assaults from
the
environment,
yet
it
remains
surprisingly intact.

Day after day, our DNA is damaged by UV radiation,

free radicals and other carcinogens, but despite of


that, our DNA at a molecular level is inherent.
Thousands of changes happen in our genome at
cellular level, but these changes affect our cells
when a process that happens thousands of times a
day are divided.
The reason why our genetic material is not
destroyed by chemical levels is due to the large
number of molecular systems that control and
repair continuously our DNA.

Nobel Prize in Chemistry 2015


The Royal Swedish Academy of Sciences has decided to award

Tomas Lindahl, Paul Modrich and Aziz Sancar the Nobel Prize in
Chemistry 2015 for their Mechanistic studies of DNA repair
Damage to the genetic material poses a threat to all organisms. To

counteract this threat, cells have evolved a series of intricate DNA


repair pathways that correct DNA lesions affecting base pairing or
structure of DNA.
Today we understand the molecular mechanisms of these pathways

in great detail, in large part due to the pioneering studies by Lindahl,


Modrich and Sancar that opened up the field.
They awarded for having mapped and explained how the cell

repairs its DNA and safeguards the genetic information.

Nobel Prize in Chemistry 2015

Background
The human genome encodes the information needed to

create a complete human being.


During every cell division, more than 3 billion DNA base
pairs are replicated and copies of the genome are
transferred to the daughter cells.
The DNA replication machinery responsible for this task
still makes occasional mistakes.
Given the size of the human genome and the large number
of cells in a human body (about 3.7 1013) mistakes will
inevitably accumulate during the lifetime of an individual.
Most of these errors will remain silent, but they can also
cause serious diseases.

Background
Despite its essential role in storing genetic

information, the DNA molecule has limited


chemical
stability
and
is
subject
to
spontaneous decay.
Processes such as hydrolysis and oxidation
occur at significant levels in vivo, in part due
to
reactive
metabolites
continuously
generated in various physiological processes.
In addition, external factors like radiation and
genotoxic chemicals will further stimulate of
DNA damage formation.

Background
The inherent instability of DNA constitutes both an opportunity

and a threat.
DNA lesions can block important cellular processes such as
DNA replication and transcription, cause genome instability
and impair gene expression.
Lesions can also be mutagenic and change the coding capacity
of the genome, which can lead to devastating diseases and
conditions associated with genome instability, including
cancer, neurodegenerative disorders and biological ageing.
Furthermore, mutagenic chemicals and radiation can also have
a healing effect; they can for instance be used to treat cancer,
by introducing DNA lesions that halt cell proliferation and
stimulate programmed cell death.

The cell has developed ways to counteract

DNA lesions and to keep DNA mutations at a


tolerable level.
A number of different DNA repair
mechanisms correct lesions and safeguard the
integrity of the genome.
Four fundamental DNA repair pathways
delineated by this years Nobel Prize laureates
will be discussed here.

Life exists so DNA must be


repairable
How

stable is DNA, really?, Tomas


Lindahl started wondering towards the end of
the 1960s.
At the time, the scientific community believed
that the DNA molecule the foundation
of all life was extremely resilient;
anything else was simply out of the question.
Evolution does require mutations, but only a
limited number per generation.

If genetic information were too unstable no multi-cellular

organisms would exist.


During his postdoc at Princeton University, USA, Tomas
Lindahl worked on the RNA molecule, a molecular
cousin to DNA.
It did not go well. In his experiment he had to heat RNA,
but this inevitably led to the molecules rapid degradation.
It was well known that RNA was more sensitive than DNA,
but if RNA was destroyed so quickly when subjected
to heat, could DNA molecules really be stable for a
lifetime? This question took hold in Lindahls mind.

He began to look for an answer to that question, and

some straightforward experiments proved that his


suspicions were correct: DNA underwent a slow
but noticeable decay.
Lindahl estimated that there were thousands of
potentially devastating injuries to the genome every
day, a frequency that was clearly incompatible with
human existence on Earth.
His conclusion was that there must be molecular
systems for repairing all these DNA defects and,
with this idea, Tomas Lindahl opened the door on an
entirely new field of research.

Special enzymes remove damage in DNA


Bacterial

DNA which, just like human DNA, consists of


nucleotides with the bases adenine, guanine, cytosine, and
thymine.
One chemical weakness in DNA is that cytosine easily loses an
amino group, which can lead to the alteration of genetic
information.
In DNAs double helix, cytosine always pairs with guanine, but
when the amino group disappears, the damaged remains tend
to pair with adenine.
Therefore, if this defect is allowed to persist, a mutation will
occur the next time DNA is replicated.
Lindahl realized that the cell must have some protection against
this, and was able to identify a bacterial enzyme that removes
damaged remains of cytosines from DNA.

Base excision repair


Bit by bit, Lindahl pieced together a molecular image

of how base excision repair functions.


Base excision repair also occurs in human beings and
Lindahl managed to recreate the human repair
process in vitro.
The decisive factor for Tomas Lindahl was the
realisation that DNA inevitably undergoes change,
even when the molecule is located in the cells
protective environment.
However, it had long been known that DNA can be
damaged by environmental assaults such as UV
radiation.

Base Excision Repair


a. DNA

glycosylase
recognizes damaged
base

b. Removes base leaving

deoxyribose sugar
c. Thereby producing an

abasic
or
AP
(apurinic/
apyrimidinic) site by
base flipping out
d. AP endonuclease cuts

phosphodiester
backbone
e. DNA

polymerase

Aziz Sancar - investigating how cells


repair UV damage
Bacteria have two systems for repairing UV damage:
light-dependent photolyase,
a second system that functions in the dark

That had been discovered using three UV-sensitive strains of bacteria


which carried 3 different genetic mutations: uvrA, uvrB and uvrC.
As in his previous studies of photolyase, Sancar began investigating

the molecular machinery of the dark system.


He had identified, isolated and characterized the enzymes coded by
the genes uvrA, uvrB and uvrC.
In ground-breaking in vitro experiments he showed that these enzymes
can identify a UV-damage, then making two incisions in the DNA
strand, one on each side of the damaged part. A fragment of 12-13
nucleotides, including the injury, is then removed.

Similar mechanisms for UV damage


repair in humans and bacteria
Aziz Sancar mapped the next stages of nucleotide excision

repair.
In parallel with other researchers, including Tomas Lindahl,
Sancar investigated nucleotide excision repair in humans.
The molecular machinery that excises UV damage from
human DNA is more complex than its bacterial counterpart
but, in chemical terms, nucleotide excision repair functions
similarly in all organisms.
Sancar eventually returned to the enzyme, photolyase
uncovering the mechanism responsible for reviving the
bacteria. In addition, he helped to demonstrate that a
human equivalent to photolyase helps us set the circadian
clock.

Nucleotide excision repair


Recognizes bulky lesions that block DNA

replication (i. e. lesions produced by carcinogens)-example, UV pyrimidine photodimers


Common distortion in helix
Incision on both sides of lesion
Short patch of DNA excised, repaired by
repolymerization and ligation
In E. coli, mediated by UvrABCD. Many more
proteins involved in eukaryotes can be coupled to
transcription (TCR, transcription coupled repair)
Defects in NER underlie Xeroderma pigmentosum.

Nucleotide Excision Repair


(in E. coli of a T-T dimer)

Endonuclease cuts on either side of


damage (~20 nt altogether).
Strands unwound by helicase.
UvrC has the endonuclease activity;
UvrA and B bind to the damaged site.
NER can also repair other types of DNA
damage.
Essential in humans; people with
Xeroderma pigmentosum have reduced
NER & extreme UV sensitivity. Also, a
NER deficiency in humans promotes
cancer. Probably a minor pathway for
dimer repair in organisms with
photolyases.

1. UvrA,B
2. UvrC

3. UvrD

It pays off to learn about DNA


stuff
Paul Modrich
DNA stuff really central to Paul Modrichs

life. He examined a series of enzymes that


affect DNA: DNA ligase, DNA polymerase and
the restriction enzyme Eco RI.
When he subsequently, shifted his attention
to the enzyme Dam methylase he stumbled
over another piece of DNA stuff that would
come to occupy him for a large part of his
scientific career.

Interweaving two strands of


research
Dam methylase couples methyl groups to DNA.

These methyl groups could function as


signposts, helping a particular restriction
enzyme to cut the DNA strand at the correct
location.
However, there was a different signalling
function for the methyl groups on DNA.
A bacterial virus with several occurrences of
mismatching bases in the DNA was constracted.
For instance, A could be placed opposite C,
instead of T.

When these viruses infect bacteria, the bacteria

corrected the mismatches.


Among other things, that it could be a repair
mechanism that corrected the faulty matches
that sometimes occur when DNA is replicated.
Perhaps the methyl groups on the DNA helped
the bacteria identify which strand to use as
template during correction.
As the new DNA strand, the faulty replica, was
still unmethylated, maybe that was how it could
be identified and corrected?

The methylation of DNA Modrichs and Meselsons

worked together, they created a virus with a number of


mismatches in its DNA.
This time, Modrichs dam methylase was also used to
add methyl groups to one of the DNA strands.
When these viruses infected bacteria, the bacteria
consistently corrected the DNA strand that lacked
methyl groups.
Modrich and Meselsons conclusion was that DNA
mismatch repair is a natural process that corrects
mismatches that occur when DNA is copied, recognising
the defect strand by its unmethylated state.

DNA mismatch repair


The DNA replication machinery is not error-free.

There is always the possibility that an incorrect


nucleotide is introduced during synthesis of a
new DNA strand.
These types of errors are known as mismatches
and they have the capacity to change the
sequence of DNA, i.e. to introduce mutations.
As a first line of defence against mismatches,
replicating DNA polymerases contain a 3 to 5
exonuclease activity that allows them to
proofread the newly synthesized DNA strand.

DNA mismatch repair


The exonuclease activity can correct mistakes

during DNA replication by reversing the


direction of the polymerase and excising
incorrectly introduced nucleotides.
Even if proofreading efficiently corrects most
mistakes made during DNA synthesis, some
non-Watson-Crick base pairs still remain. To
correct these errors, cells use mismatch repair.

DNA mismatch repair


Today we know that all but one out of a

thousand errors that occur when the human


genome is copied, are corrected by mismatch
repair.
However, in human mismatch repair, we still
do not know for sure how the original
strand is identified.
DNA methylation has other functions in our
genome to that of bacteria, so something else
must govern which strand gets corrected
and exactly what remains to be clarified.

Mismatch repair (MMR)


Despite extraordinary fidelity of DNA synthesis, errors do

persist
Such errors can be detected and repaired by the postreplication mismatch repair system
Prokaryotes and eukaryotes use a similar mechanism
with common structural features
Defects in MMR elevate spontaneous mutation rates 101000x
Defects in MMR underlie human predisposition to colon
and other cancers (HNPCC)
MMR also processes mispairs that result from
heteroduplex DNA formed during genetic recombination:
act to exclude homeologous recombination

Mismatch Repair
Problem: how do cells know which is the right

template strand?
In E. coli, new DNA not methylated right away

Mismatch recognized by mutS, then mutL binds and

attracts mutH (endonuclease that cleaves mismatch


and nearest CTAG that is not methylated)

Eucaryotes (including Arabidopsis) have mutS

and mutL homologues, but no mutH

Also have the requisite exonucleases, but not clear

how the strand specificity is determined

Mismatch Repair
In E.coli, A of each
GATC is methylated.

Dam methylase
is delayed for
about 10 min,
then it
methylates the
new replicon.

mutH is
endonuclease

Summary
Tomas Lindahl, Paul Modrich, and Aziz Sancar have made fundamental

and groundbreaking discoveries on the enzymatic mechanisms of


DNA repair.
Lindahl demonstrated that DNA is an inherently unstable
molecule, subject to decay even under physiological
conditions.
Guided by this observation, Lindahl identified a completely new
group of DNA glycosylases and described their role in base
excision repair.
Modrich transformed the field of mismatch repair from genetic
observations to a detailed biochemical understanding, first in bacteria,
and later in eukaryotic cells.
Sancar has transformed the field of nucleotide excision repair, from
genetics and phenomena in cell extracts, to a detailed molecular
description of the mechanisms involved, first in bacteria, and later also
in eukaryotic cells.
Sancar also explained the molecular mechanisms underlying
photoreactivation, the first form of DNA repair described.