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Transcript
DNA Repair and
Recombination
Mutations
• A mutation is any permanent change in the DNA nucleotide sequence.
• Simplest form of mutation: a nucleotide substitution, where one nucleotide is replaced
by a different one.
• Many mutations occur in non-gene areas of the DNA and have very little
or no effect on the organism.
• Only about 1.5% of human DNA is contained in genes.
• In bacteria, 80% or more of the DNA is in genes.
• Mutations in genes can alter or destroy the function of the gene product.
• A closely related issue: DNA damage, which can stop DNA replication or
cell division
• DNA polymerase may not be able to use a non-standard nucleotide as a
template.
• Single stranded breaks in the DNA molecule will have trouble during
DNA replication
• Double-stranded breaks are worse: the two ends of the broken DNA
need to be re-assembled before cell division or replication can succeed.
• There are several mechanisms the cell uses to detect and repair DNA
damage before it become permanent.
Somatic vs. Germ Line Mutations
• An important distinction: germ line mutations
vs. somatic mutations.
• germ line mutations occur in the cells that become
sperm or eggs (= gametes). Germ line mutations
heritable: they can be passed on to future
generations.
• On the other hand, somatic mutations occur in other
body cells, and can only affect the individual, not any
descendants.
• Most somatic mutations have little effect on the
individual. However, cancer is caused by certain
specific somatic mutations.
• This distinction mostly applies to animals. In the
early embryo, animals set aside a small group of
cells to become the germ line.
• In plants, any cell has a chance of becoming a gamete.
• Single cell organisms don’t use a germ line either.
Mutations and Evolution
• Mutations are the raw material of evolution. Species
change over time due to the effects of natural selection
on organisms with different mutations.
• Most mutations either have no effect or a deleterious
effect on the fitness of an organism (fitness = ability to
survive and reproduce).
• Occasionally a mutation increases an organism’s fitness.
Natural selection causes the frequency of these genetic
alleles in the population.
• Sometimes a mutation has no effect on fitness, or even a
slightly bad effect, but a change in the environment
makes the mutation valuable. This is called a preadaptive mutation or exaptation.
• Mutations can cause a lot of harm to an organism. Thus
it is important to minimize the number of mutations. It
is not possible to prevent them, but it is possible to
repair the damage they cause.
The chart above shows the effect of various
mutations on vesicular stomatitis virus. The
unmutated virus has a fitness of 1.0. Almost
half of the mutations give a fitness of 0 (=
dead), and most of them are deleterious
(fitness less than 1.0). A few mutations have
a fitness greater than 1 (i.e., they are better
than the original).
Mutations vs.
DNA Damage
• The mutation process starts with DNA damage (or replication
errors), but only becomes an actual mutation if the damage is not
repaired.
• Initially, the damage or mis-incorporated base causes a
mispairing in the DNA double helix: there is something other than
an A-T or G-C base pair.
• Many of these mispairings are identified and repaired by the cell.
There are enzymes that detect any spots on the DNA that are not AT or G-C base pairs.
• The damage becomes a mutation only after the mispaired bases
are replicated. This converts the mispaired bases into regular A-T
and G-C base pairs that look completely normal to the cell’s
enzymes.
• The mutant bases are now permanently part of the DNA, inherited
in all future cell generations.
Sources and Types of Mutation
• Internal sources:
• sometimes DNA polymerase makes a mistake, despite the proofreading
function
• Interactions between DNA and other molecules in the cell, including water
and reactive oxygen species.
• External sources
• Ultraviolet light and other ionizing radiation
• Reactive chemical compounds
• Changes in a single nucleotide
• Breaking the DNA backbone, either a single stranded break or a
double stranded.
DNA Polymerase Errors
• As we discussed in the Replication lecture,
DNA polymerase sometimes incorporates
the wrong nucleotide.
• Often due to the tautomeric shift, where
movement of a Hydrogen atom puts the base
in an unusual configuration that allows
mispairing.
• DNA polymerase detects many of these
errors as soon as they occur.
• At which point the DNA polymerase uses its 3’
→ 5’ exonuclease activity to remove the new
base and try again.
• The process of detected and correcting these
mismatches is called proofreading.
• Some errors still occur, and need to be
corrected.
Spontaneous Depurination and Deamination
• Some DNA damage is simply the result of
the aqueous environment. H2O
spontaneously dissociates into H+ and OH-,
which are reactive.
• The bond between a purine base and
deoxyribose is prone to being hydrolyzed
by H+ ions. This is depurination, and it
leads to an apurinic site. DNA polymerase
usually skips over the apurinic site,
producing a 1 base pair deletion in the
daughter DNA molecule.
• The amino group on the cytosine base is
also easily lost by hydrolysis. This is
deamination, and it converts cytosine into
uracil. Uracil pairs with adenosine, so
after replication, a C-G base pair has been
converted to an A-T base pair.
• Note that uracil is not a DNA base.
• Amino groups on other bases (e.g. guanine)
can also be lost, converting the base to
something non-standard.
Oxidative Damage and Alkylating Agents
• Aerobic metabolism in the mitochondria uses oxygen. Some of
this oxygen gets converted to reactive oxygen species, primarily
hydrogen peroxide (H2O2) and superoxide (O2-)
• Reactive oxygen species can attack bases, as well as break the DNA
backbone.
• They also cause other forms of damage in the cell.
• Many chemical compounds can attack various bases and attach
bulky alkyl (hydrocarbon) groups.
• DNA polymerase has a hard time processing these modified bases.
• Tobacco smoke contains benzo(a)pyrene, which is modified by the
liver to produce a reactive derivative.
• This derivative attacks purines and adds a bulky group that does not
form proper base pairs.
UV-Induced Thymine Dimers
• Two T’s next to each other in the DNA sequence are stacked on top of
each other in the actual DNA molecule. UV light can link them together
to form a thymine dimer. This is a common effect of sunlight.
• Thymine dimers distort the DNA double helix, and cause DNA
polymerase to stall, so replication is blocked.
• If not repaired, thymine dimers can cause various forms of skin cancer.
• The genetic disease xeroderma pigmentosum is caused by the failure to
repair thymine dimers. People with this disease develop freckles very
easily, some of which turn cancerous. To avoid this, they need to stay out
of direct sunlight.
Repair Mechanisms
• There are several different systems
in the cell to repair different forms of
damage to one strand of the DNA.
However, most of them work in the
same basic fashion:
1. Locate the damaged region
2. Excise the damaged and the
surrounding nucleotides. This creates
a free 3’ OH group that DNA
polymerase can build off of.
3. Use a special DNA polymerase to resynthesize the damaged strand
4. Seal the nick remaining after DNA
polymerase with DNA ligase.
Mismatch Repair
• DNA polymerase with its proofreading mechanism still
makes about 1 error per 107 bp. (The human genome is
about 3 x 109 bp).
• The mismatch repair system fixes about 99% of these
errors, giving an error rate of 1 in 109 bp.
• It works like other DNA repair mechanisms. The initial
step, recognizing the mismatch, occurs because
mismatched bases form a slight bulge in the DNA
double helix.
• The tricky part is recognizing which strand still has the
correct base and which has the incorrect base.
• The incorrect base is on the newly synthesized strand
• Current thought is that newly synthesized DNA has nicks in
it, both in the lagging strand (between Okazaki fragments)
and in the leading strand (presumably because DNA
polymerase falls off the DNA and needs to be restarted).
• The mismatch repair system recognizes which strand has
nicks, and repairs it with the information from the other
strand.
Base Excision Repair
• Many forms of DNA damage occur
repeatedly; a good example is
deamination of thymine to form
uracil. These sites are easily dealt
with by the base excision mechanism.
• The cell contains several DNA
glycosylases, which detect specific
kinds of altered bases and cleaves
the bond between the base and
deoxyribose. This creates an abasic
site.
• Next, AP endonuclease cuts out the
abasic nucleotide. This creates a
short gap with a free 3’ –OH.
• A special DNA polymerase resynthesizes the DNA in the region,
and then DNA ligase seals the final
nick.
Nucleotide Excision Repair
• Nucleotide excision repair works on DNA
damage that none of the base excision
repair glycosylases recognize: bulky alkyl
groups attached to a base, thymine
dimers, etc.
• This system recognizes warps and bulges
in the DNA double helix, then excises 1020 bases on either side of the problem,
then uses DNA polymerase to
resynthesize the second strand, and finally
seals the remaining nick with DNA ligase.
Double Stranded Breaks
• If both strands of DNA break, there is no easy way
to repair it: the ends just float freely away from
each other.
• One important mechanism of repairing double
stranded breaks is non-homologous end joining.
• Proteins bind to the broken DNA ends, then clean
them by filling in single stranded gaps and making
sure each 3’ end has a free –OH and each 5’ end
has a free PO4 on it. The ends are then ligated
together.
• One of the proteins that binds to the broken
chromosome ends in BRCA1. Mutations in this
gene are a major cause of breast cancer.
• If there are more than one pair of broken ends in
the cell, this mechanism can easily join the wrong
ones together. This is a major cause of
chromosomal rearrangements like inversions and
translocations.
Repair by Homologous Recombination
• A second way to repair double stranded breaks is to use the same
mechanism used to perform crossing over in meiosis: homologous
recombination. We will discuss crossing over later, but right now, we
will examine how this process can repair a break.
• The key is, in a diploid there are 2 copies of every sequence, one on
each of the homologous chromosomes. If one chromosome is
broken, you can use the information on the other one to repair it.
• The first step is exonuclease activity that trims back the 5’ ends on
both sides of the break, creating long single stranded ends with free
3’ -OH groups.
• Next, the key step, strand invasion: the broken end of one DNA
strand invades the unbroken double helix of the homologous
chromosome and base pairs with it.
• This broken end acts as a primer and a DNA polymerase extends it.
• After some DNA synthesis, the elongated broken end is long enough
to invade the other broken piece of DNA, and more DNA polymerase
activity resynthesizes all the bases in the broken region.
• DNA ligase then seals up the nicks.
Steps in DNA Repair by Homologous Recombination, 1
1. Double stranded break in one homologue.
Note that the bottom (blue) DNA molecule is
written with the 3’->5’ strand on top, which
is opposite of the usual way of writing it.
2. Cutting back of 5’ ends by an exonuclease,
producing long tails with free 3’ ends.
3. Strand invasion: one 3’ end invades the
double helix of the homologous DNA.
4. The strand that is displaced forms a Dloop (displacement loop)
Steps in DNA Repair by Homologous Recombination, 2
5. The invading 3’ end is elongated by DNA polymerase,
using the homologue as a template. The D-loop
expands.
6. Capture of the other 3’ end by unpaired portion of
the D-loop.
7. The captured 3’ end is elongated by DNA
polymerase.
8. Ligation of free ends, creating 2 Holliday junctions
(places where 2 DNA molecules joined together).
9. Resolution of the Holliday junctions by horizontal
cuts and religation
10. Note that some sections of both DNA molecules are
heteroduplex and thus possibly having some mispaired
bases that will need to be repaired or resolved by
replication.
11. Note that part of the broken DNA molecule has
been converted to the sequence from the unbroken
DNA molecule, with that area of the broken DNA lost.
Homologous Recombination
• Homologous recombination is the process by which 2 nearly
identical DNA molecules break and rejoin to form new hybrid
molecules. It is also called crossing over.
• Homologous recombination is found in almost all organisms,
in all 3 domains of life. It may have evolved from double
strand break repair.
• Homologous recombination creates new combinations of
genes, which increases the chances of having a combination
that is more evolutionarily fit than either parent.
• In eukaryotes it occurs during meiosis (prophase of meiosis 1,
for those who have taken my BIOS 308 class). In prokaryotes
it can occur at any time.
• The mechanism is the same as the homologous
recombination repair mechanism, except for the very end.
Homologous Recombination Mechanism,1
• Crossing over
starts with a
double stranded
break, catalyzed
by an enzyme
(instead of a
random event as
in DNA repair).
• The initial steps of
recombination are
identical to repair
by homologous
recombination.
1. Double stranded break in one
homologue.
2. Cutting back of 5’ ends by an
exonuclease, producing long tails with
free 3’ ends.
3. Strand invasion: one 3’ end invades the
double helix of the homologous DNA.
4. The strand that is displaced forms a Dloop.
5. The invading 3’ end is elongated by DNA
polymerase. The D-loop expands.
6. Capture of the other 3’ end by unpaired
portion of the D-loop.
7. The captured 3’ end is elongated by DNA
polymerase.
Homologous Recombination Mechanism,2
• Outside genetic markers
A and B (on the red
DNA) and a and b (on
the blue DNA) have
been added to make the
results clearer.
• However, there is a
critical difference at
resolution of the
Holliday junctions.
Rather than cutting both
junctions horizontally, at
the place where the
strands cross, one
junction is cut vertically
(9A)
8. Ligation of the newly synthesized
strands to the old strands.
9. Horizontal cut at one Holliday junction.
9A. Vertical cut at the other Holliday
junction.
10A. The cut ends are ligated. The
strands cross in this 2-dimensional
drawing, so that the proper joins are made
between 5’ and 3’ cut ends.
11A. After the DNA molecules are
separated, it is clear that recombination
has occurred: A on the red DNA
(chromosome) is attached to b from the
blue chromosome, and a from the blue is
attached to B from the red.
Holliday Junctions and the Results of Homologous
Recombination
• Two Holliday junctions appear during homologous
recombination. They need to be resolved by cutting 2
strands and then ligating the cut ends so that the two
DNA molecules can separate from each other.
• The recombination of genetic markers outside the
recombination site only occurs if one cut is horizontal and
the other is vertical (as shown in the previous slide).
• If both cuts are horizontal, or both are vertical, the
outside markers (A, B, a, b) are not recombined: they stay
linked together. Only a short region at the site of the
recombination is heteroduplex.
• If this happens within a gene, that gene can undergo gene
conversion: it can switch from one allele (say, the red allele) to the
other (the blue allele) without any recombination of markers
outside the recombination site.
• Gene conversion is rare, because most recombination occurs in
DNA regions not part of any gene.
In this picture of a Holliday junction and
its resolution, what happens to the
crossed strands is made easier to see by
rotating the lower part of the junction.