BIOL 200 Lecture Notes - Rad51, Transcription Factor Ii H, Guanine
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Naveen Sooknanan McGill Fall 2011
DNA Repair and Recombination:
Over the course of evolution, errors in DNA replication can introduce permanent, transmissible
changes into a daughter cell of organism. This is known as a mutation to DNA. This causes the
encoding for different mRNA molecules after translation, and, eventually, different amino acids
coded during translation.
Mutagens, on the other hand, are chemical compounds, ultraviolet radiation, or ionizing
radiation (X-rays and atomic particles) which can increase the frequency of mutation.
A carcinogen is an agent that causes cancer. All carcinogens are mutagens. Carcinogens can
introduce the following changes into a normal cell:
Self-sufficiency in growth signals
Insensitivity to antigrowth signals
Evasion of apoptosis
Limitless replicative potential
Tissue invasion and metastasis
There are multiple methods of DNA repair through which the cell can prevent the occurrence of
mutations. Each of the mechanisms can take place at different times after transcription in order to
maximize effectiveness. Note that we will use a eukaryotic model to explain DNA repair.
E. coli DNA polymerase can introduce 1 error in every 10,000 nucleotides incorporated
into new polynucleotide strands. An error incorporation rate like this would cause too
many mutations to allow for our survival.
Due to the effectiveness of DNA repair mechanisms, the actual rate of error incorporation
is in fact 1 in every 1,000,000 nucleotides after measures of correction.
Some DNA polymerases have a proofreading activity which reads the newly synthesized strand
in the 5’ 3’ direction.
DNA polymerase δ, and not α, is able to read the new
strand with an exonuclease activity
o This is why Pol δ takes over for Pol α in DNA
When there is a mistake, Pol δ chews off the 3’ end of the
new strand and repolymerizes it with the correct base pairing.
Another mechanism, called base excision repair is able to fix errors after replication. It is used to
fix errors caused by mutagens.
De-amination of cytosine into uracil can occur spontaneously in the cell, for example
during cell metabolism
o This is not good if it happens in DNA and must be repairs by base excision repair
Cytosine can also be methylated on carbon 5 via and enzyme called DNA
o This is not necessarily a bad thing, in fact it is necessary for gene regulation,
transposon silencing and chromatin remodelling (gene compacting)
o Methylated cytosine can also be transformed into thymine by de-amination
Naveen Sooknanan McGill Fall 2011
Base excision repair undergoes a multitude of steps involving gap repair in order to repair one
nucleotide in a polynucleotide strand.
DNA glycosylase hydrolyzes the bond between the
mispaired base and the sugar phosphate backbone, leaving
the backbone attached to the sequence and removing only
the T in this example
Apurininc/Apyrimidinic Endonuclease 1 (APE1) cuts the
sugar phosphate backbone at the 5’ end
AP lysase associates with DNA Polymerase β cuts the 3’
end of the sugar phosphate backbone (deoxyribose
phosphate), removing it from the sequence
The new, correct nucleotide is brought in as a dNTP and is
synthesized into the sequence by DNA polymerase β and the sugar phosphate backbone
is ligated by DNA ligase (called gap repair).
Mismatch excision repair is coupled with DNA replication; it happens shortly after replication
takes place. This mechanism is used on newly synthesized strands where errors were
incorporated and missed by proofreading.
Once an error is introduced, a series of steps is taken out to carry out mismatch excision repair.
This mechanism also involves gap repair.
Proteins MSH2 and MSH6 bind to the daughter strand and associate with the erroneous
o It’s not known how these complexes recognize which is
the newly synthesized strand, but it must be able to in
order for this mechanism to work
Endonuclease MLH1 and PMS2 bind to the complex and are
DNA helicase and DNA exonuclease then unwind and digest
the area around the erroneous base.
Pol δ then synthesizes the removed area of the daughter strand,
replacing the erroneous base with the correct one. DNA ligase
then repairs the sugar phosphate backbone. This again is an
example of gap repair.
Nucleotide excision repair modifies erroneous bases caused by
post-transcriptional changes introduced by mutagens.
UV radiation and chemical compounds can cause a
distortion in the normal shape of DNA. This is what
allows various enzymes to recognize the error in the sequence of DNA. This can cause
thymine-thymine dimers which involves the covalent bonding of adjacent thymine bases.
There errors are usually passed over by DNA proofreading.
Naveen Sooknanan McGill Fall 2011
Enzymatic modification of Aflatoxin B, a contaminant in grain produce
and peanut butter, causes it to bond to nitrogen 7 of guanine which
causes a massive shape distortion
The mechanism for nucleotide excision repair is slightly different from the two described above,
but it also involved gap repair.
The irregular shape in the double helix is recognized by the protein
XP-C which associates with the protein 23B
o XP-C opens up the structure to produce a bubble
A transcription factor TFIIH binds to the distorted strand and an
RPA binds to the opposite side. An endonuclease called XP-G
then cuts the erroneous strand only on the 3’ end. Another
endonuclease called XP-F then cuts the other side of the mutation
DNA polymerase (maybe epsilon, not well known) then
synthesizes the DNA re-synthesizes the mutated strand with no
mutations and DNA ligase repairs the sugar phosphate backbone
When both strands of the DNA molecule are broken, there is a potential loss of information in
the gene. This can either be repairs by double strand break repair by end joining or double strand
break repair by homologous recombination.
Mutagens such as ionizing radiation (X-rays) or anticancer drugs like bleocymin can
cause single or double stranded breaks in the DNA molecule. This causes genomic
rearrangements or translocations such as “jumping genes”. These must be repaired by
either double stranded break repair mechanism as described above.
These mutations can be especially deadly due to a loss of information from hitting a gene
Double strand break repair by end joining is an error-prone way to quickly fill the gaps of a
double stranded break to prevent further loss from exonucleases. It can be
seen as a “hail Mary” or last resort mechanism to minimize information
A complex formed by DNA-dependant protein kinase (DNA-PK)
and KU80/KU70 heterodimer bind to the break ends
The overhangs are digested by exonucleases and the blunted ends
are ligated together
This mechanism can actually introduce mutations to the sequence, but the loss of a few
nucleotides if far less severe than having the whole DNA molecule digested by exonucleases.
Double strand break repair by homologous recombination is an error free method of DNA repair
which uses a full, undamaged duplex as a template to repair a duplex with a double strand break.
The ends of the broken DNA duplex are digested in order to leave 3’ sticky ends
A strand invasion from the intact to broken DNA duplex is mediated by RecA in
prokaryotes or Rad51 in eukaryotes
o The intact strand is used as a template to produce Watson Crick base pairs which
contain all genetic information