This summary contains information about: DNA replication mechanisms, DNA polymerase III proofreading, strand-directed mismatch repair, post-replication DNA repair, base excision repair, mutations, nucleotide exchange repair, double-strand break repair, nonhomologous end joining, homologous recombin...
Experimental Cell Biology: Cellular Stress Responses
DNA replication mechanisms DNA repair-associated syndromes
DNA templating is the mechanism the cell uses
to copy the nucleotide sequence of one DNA
strand into a complementary DNA sequence.
This process requires the separation of the
DNA helix into two template strands. In the
replication fork, the DNA gets polymerized in
the 5’ -> 3’ direction (leading strand). On the
other strand are Okazaki fragments growing
in the 5’-to-3’ direction using DNA primase to
make short RNA primers, and are later joined Proof-reading by DNA polymerase III
together by DNA ligase (lagging strand, the
polymerization is the opposite to the direction
of the DNA chain growth). Sometimes a wrong
base pair gets built into the DNA. Luckily DNA
polymerase has a proofreading system and
double-checks the base-pair geometry before it
catalyzes the addition of the next nucleotide.
The next error-correcting reaction is known as
exonucleolytic proofreading. This takes place
immediately after those rare instances in
which an incorrect nucleotide is covalently Strand-directed mismatch repair system in
added to the growing DNA chains. DNA eukaryotes. The newly
polymerase molecules correct this mismatch synthesized lagging-
in the 3’-to-5’ proofreading exonuclease. strand DNA contains
nicks before the strand
is sealed by DNA ligase
(Okazaki fragments).
This is also how you
can distinguish the
a new strand from the
old. The nicks, also
called single-strand
breaks, provide a signal
that directs the
mismatch proofreading
system to the appropriate strand. The
importance of mismatch proofreading in humans
All this cellular stress can eventually lead to is seen in individuals who inherit one defective
DNA damage. DNA damage can then lead to: copy of a mismatch repair gene. These people
• Mutations that affect protein function have a marked predisposition for certain types of
(misfolded/unfolded proteins) cancers. DNA gets methylated shortly after
• Cell death as a result of blocked replication replication. The mismatch repair mechanism
• Transformation of the cell into a cancer cell recognizes the wrongful replicated DNA because
it is not methylated yet. MutS localizes the
And despite all these safeguards against DNA damage. MutL protein scans the surrounding
replication errors, DNA polymerases DNA for a nick. At the nick will be flexibility
occasionally makes mistakes. Therefore cells because one of the two strands is not intact.
have another ways to correct these errors and Nicks occur at sites where replication has
cope with the damage. These processes are: finished but the strand has to be joined again
• Strand-directed mismatch repair with the rest of the DNA. Mutual connects the
• DNA repair (SOS) response site of the damage with the site of the nick. Then
• Protein folding control (heat-shock the strand can be removed. DNA ligase will
response) attach the DNA again.
, When the cell experiences heavy damage, the Cells have multiple pathways to repair their DNA
cell will use extra DNA polymerases, called using different enzymes that act upon different
translesion DNA polymerases. Humans have 7 kinds of lesions:
of this translesion DNAP. These DNAPs can • Base excision repair (BER)
build in nucleotides and restore DNA structure • Nucleotide exchange repair (NER)
and replications. The two pathways differ in the way in which they
remove the damage from the DNA.
Base excision repair
This pathway involves a
battery of enzymes called
DNA glycosylases. Each of
these enzymes can recognize
a specific type of altered base
in DNA and catalyze its
hydrolytic removal. This
type of repair can repair up
to 6 types of damages. When
the H-bond pattern of a
basepair is changed/dis-
rupted an enzyme mediates
“flipping-out” (this leads to
excision) from the helix
Post-replication DNA repair so that the DNA glycosylase
Deamination and depurination are chemical can probe all faces of the base. Once the enzyme
reactions that create DNA damage in cells. finds the damaged base, it removes that base
Depurination can release guanine and adenine from its sugar. The “missing tooth” created is
from DNA. A deamination reaction converts recognized by an enzyme called the apurinic/
cytosine to an altered DNA base. These apyrimidinic endonuclease (AP) enzyme. AP
reactions take place in double-helical DNA. A cuts the phosphodiester backbone, after which
result of UV radiation is the type of damage that the resulting gap is repaired. Depurination also
causes a thymine dimer (covalent linkage leaves a deoxyribose sugar with a missing base,
between tho adjacent pyrimidine bases in DNA). these are directly repaired beginning with AP
If left uncorrected when the DNA is replicated, endonuclease, following the bottom half of the
most of these changes lead to the deletion or pathway.
more base pairs to a base-pair substitution in
the daughter DNA chain. UV-induced mutations
UV cross-links two
nucleotides which
creates pyrimidines
Nucleotide Exchange repair
This mechanism can repair the damage that
caused pyrimidine dimers (T-T, T-C and C-C). In
this pathway, a large multienzyme complex
scans the DNA for a distortion in the double
Deaminated nucleotide/depurinated helix, rather than for a specific base change.
Once it finds a lesion, it cleaves the
phosphodiester backbone of the abnormal strand
on both sides and a DNA helicase peels away the
single-strand oligonucleotide containing the
lesion. The gap is repaired by DNA polymerase
and DNA ligase.
C, G, A or T substitution/A, G or T deletion
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