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DNA repair
Of the thousands of random changes created every
day in the DNA of a human cell by heat, metabolic
accidents, radiation of various sorts, and exposure
to substances in the environment, only a few
accumulate as mutations in the DNA sequence.
DNA repair is essential for all
organisms.
• Because the survival of the individual
demands genetic stability, analysis of the
genomes of bacteria and yeasts has revealed
that several percent of the coding capacity
of these organisms is devoted solely to
DNA repair functions.
What causes DNA damage?
•
•
•
•
Thermal fluctuations
Ultraviolet radiation
Alkylating agents
Gamma rays and X-rays
DNA damage delays progression
of the cell cycle
• Cells will delay progression of the cell cycle
until DNA repair is complete.
• For example, E. coli sulA protein is an
inhibitor of cell division and it is expressed
in response of SOS signal.
• Human ATM protein is a protein kinase that
will send signals in response of oxygeninflicted DNA damage.
Thermal fluctuations result in
depurination of A and G
• Depurination (hydrolysis of the N-glycosyl
linkages to deoxyribose) of A and G happen about
5,000 times every day per cell (in total).
Thermal fluctuations also cause
deamination of cytosine
• Deamination of cytosine produces uracil, a base that will
not appear in DNA.
• Deamination happens about 100 times every day per cell.
Deamination
also happens to
other bases
• Deamination of A
(producing
hypoxanthine) and
G (producing
xanthine) produces
bases that will not
occur in DNA
naturally, therefore
they will be easily
recognized.
Ultraviolet radiations
• Ultraviolet radiations can produce a
covalent linkage between two adjacent
pyrimidine bases in DNA to form
pyrimidine dimers.
Thymidine dimers
• Thymidine dimers are
one of the products
produced on DNA
irradiated by ultraviolet
radiation.
Alkylation
• Alkylating agents like EMS (ethylmethane
sulfonate) are electrophiles. They seek center of
negative charge (for example, DNA) and attack
them by adding alkyl groups (carbon-containing
groups).
Results of DNA damages left
unrepaired
• Unrepaired DNA damages can lead to
deletion of one or more bases pairs (in the
case of depurination) or to a base-pair
substitution in the daughter DNA chain (in
the case of deamination and alkylation)
when they are being replicated.
Unrepaired
depurination leads
to deletion
Unrepaired
deamination leads to
base-pair substitution
Why most of the organisms have
double-stranded DNA as their
genome?
• For safe storage of genetic information,
double-stranded DNA is the best choice for
genome.
• When one strand is damaged, the
complementary strand retains an intact copy
of the same information, and the copy is
generally used to restore the correct
nucleotide sequences to the damaged strand.
Two common pathways for DNA
repair
• Base excision repair
- DNA glycosylases + AP endonucleases
- AP endonucleases
• Nucleotide excision repair
Base excision repair
• Base excision repair involves a battery of DNA
glycosylases.
• There are at least six types of DNA glycosylases:
- removing deaminated Cs and As
- removing different types of alkylated or oxidized
bases
- removing bases with opened rings
- removing bases in which a C=C has be converted
to C-C
- removing T from T-G pair (inefficiently)
Mechanisms of DNA
glycosylases action
• DNA glycosylases
flip out base while
travel along DNA to
find out the altered
nucleotide from the
helix.
Mechanisms of DNA glycosylases
action
• Once a damaged base is
recognized, the DNA
glycosylase creates a
deoxyribose sugar that
lacks the base (AP site).
After DNA glycosylases create
AP site, AP endonucleases join
• AP endonucleases
recognize the AP site,
cut the phosphodiester
backbone, leave a gap.
After AP endonucleases
• The gap is then
repaired by DNA
polymerase and
DNA ligase.
AP endonucleases are also
involved in repairing depurinated
DNA
• Depurination also
leaves an AP site.
• Depurinated DNA
will be directly
repaired by AP
endonuclease as
described
previously.
Nucleotide excision repair
• Nucleotide excision repair can repair almost
any large change in the structure of the
DNA double helix.
• A large multienzyme complex scans the
DNA for a distortion in the double helix,
rather than for a specific base change.
Nucleotide excision repair
• Once a bulky
lesion has been
found, the
phosphodiester
backbone of the
abnormal strand is
cleaved on both
sides of the
distortion.
Nucleotide excision repair
• The oligonucleotide
containing the lesion
is peeled away from
the DNA double
helix by DNA
helicase. DNA
polymerase and
ligase will repair the
large gap.
Deamination
problems
• Although most of
the deamination
products are
unnatural bases
and will be
recognized by
repair machinery…
Deamination problems
• Deamination of methylated C (which happens a lot in
inactivated genes) produces thymine, a naturally occurring
DNA.
• Although a special DNA glycosylase recognizes a
mismatched base pair involving T in the sequence T-G and
removes the T, but this enzyme is rather ineffective. As a
result, 1/3 of the single base mutations comes from these
methylated nucleotides!
Gamma rays and X-rays
• While all the other factors damage DNA by
modifying nucleotides, gamma rays and Xrays damage DNA by breaking one or both
strands.
• Single-strand breaks are easily repaired.
However, double-strand breaks will result in
chromosome degradation if left unrepaired.
Repairing double-strand breaks
• Double-strand breaks can be repaired either
by nonhomologous end-joining (NHEJ) or
homologous end-joining.
Nonhomologous end-joining
• Broken ends are
juxtaposed (placed sideby-side) and
rejoined by DNA
ligation, generally with
the loss of one or more
nucleotides at the site of
joining.
Homologous end-joining
• Homologous endjoining requires
special
recombination
proteins that
recognizes areas of
DNA sequence
matching between the
two chromosomes
and bring them
together.
Homologous end-joining
• A DNA replication
process then uses the
undamaged
chromosome
as the template for
transferring genetic
information to the
broken chromosome,
repairing it with no
change in the DNA
sequence.
Homologous end-joining
• Homologous end-joining is the major route
for repairing DNA double-strand breaks in
bacteria, yeasts, Drosophila, and also in
those organisms which little
nonhomologous end-joining is observed.
SOS response – the error-prone
bypass
• In E. coli, an excess of single-strand DNA
induce the expression of more than 15
genes. They will not only increase cell
survival after damage but also increase the
mutation rate by increasing the number of
errors made in copying DNA sequences.
This is due to the usage of low-fidelity
DNA polymerases.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
or other mutagenic treatment
UmuD’
UmuD’2C
(DNA pol V)
Why use low-fidelity DNA
polymerase?
• Because only low-fidelity DNA polymerase
can efficiently use damaged as template for
DNA synthesis, so it is employed during
SOS response.
• Human cells contain more than ten minor
DNA polymerases, many of which are
error-prone.
General recombination
General recombination is one type of
the genetic recombination.
General recombination
• General recombination is also called homologous
recombination. Homologous end-joining is
involved in general recombination.
• General recombination is essential for every
proliferating cells, because accidents occur during
nearly every round of DNA replication that
interrupt the replication fork and require general
recombination mechanisms to repair.
• Cross-over during meiosis also use the same
mechanism of general recombination.
General recombination
• General recombination creates
DNA molecules of novel
sequence because the
heteroduplex joint can tolerate a
small number of mismatched
base pairs. Usually these two
DNA molecules are not exactly
the same on either side of the
joint.
General recombination
mechanism (1)
• DNA helix is broken by special endonuclease (for
example, HO endonuclease in S. cerevisiae).
General recombination
mechanism (2)
• Limited degradation of the 5’ end is executed by
exonuclease.
• RecA from E. coli, a SSB and DNA-dependent
ATPase, is essential in this part to keep ssDNA in
extended conformation.
RecA
• RecA is not
only essential
in DNA repair
but also in
recombination.
• RecA has more
than one DNAbinding site so
it can hold a
single strand
and a double
helix together.
RecA in general recombination
• With RecA, the region of homology is identified before the
duplex DNA target has been opened up, through a threestranded intermediate in which the DNA single strand
forms transient base pairs with bases that flip out from the
helix in the major groove of the double-stranded DNA
molecule.
General recombination
mechanism (3): strand invasion
and branch migration
• The unpaired region of one of the single strands
displaces a paired region of the other single strand,
moving the branch point without changing the
total number of DNA base pairs.
RecA protein guides the direction
of branch migration
• The ATP-bound form
of RecA binds to DNA
tighter than ADPbound form.
• New RecA molecule
(RecA-ATP) are
preferentially added at
one end of the RecA
protein filament then
ATP hydrolysis
followed.
RecA protein is essential
• The homolog of RecA protein in human
(Rad51) is also essential.
• These proteins require accessory proteins to
help them functioning properly.
• Two of the hRad51 accessory proteins,
Brca1 and Brca2, are related to breast
cancer.
General recombination
mechanism (4)
• At this stage, DNA replicating machines use the 3’
end as primer to start DNA synthesis with the
pairing strand (the other DNA duplex) as template.
• Gene conversion also happens during this stage.
Gene conversion
Results of gene conversion
General recombination
mechanism (5)
• Holliday junction is
formed after the other
end is paired and DNA
synthesis is completed.
Holliday junction
• Holliday junction (cross-strand exchange) is the
key recombination intermediate.
• After it is formed, it can isomerize to the open
structure (B) or further isomerization will make
the crossed strand becoming non-crossing strand.
The open structure of Holliday
junction
• When the open
structure of Holliday
junction is formed,
two RuvB hexamer
and one RuvA
tetramer will engage
the resolution of
Holliday junction,
producing recombined
DNA molecules.
RuvA tetramer
Two RuvB hexamer
use energy from ATP hydrolysis to extend the heteroduplex region
by moving out the open circle.
General recombination
mechanism (6)
• With help of RuvA,
RuvB and other
proteins, selective
strands were cut and
ligated and exchanged
DNA with
heteroduplex region of
several thousand base
paired were formed.
General recombination leads to
different products in mitotic and
meiotic cells
Guided by
specific
proteins
99% mitotic
cells go this
way
Proteins involved in mismatch
repair is also involved in general
recombination
mutL
mutS
(mutH)