Dr Mohammad S Alanazi, MSc, PhDMolecular Biology
KSU
DNA repair: mechanisms, methods to study DNA repair, syndromes
DNA Lesions That Require RepairDNA Lesion Example/Cause
Missing baseRemoval of purines by acid and heat (under physiological conditions ≈104 purines/day/cell in a mammalian genome); removal of altered bases (e.g., uracil) by DNA glycosylases
Altered base Ionizing radiation; alkylating agents (e.g., ethylmethane sulfonate)
Incorrect base Mutations affecting 3′ → 5′ exonuclease proofreading of incorrectly incorporated bases
Bulge due to deletion or insertion of a nucleotide
Intercalating agents (e.g., acridines) that cause addition or loss of a nucleotide during recombination or replication
Linked pyrimidines
Cyclotubyl dimers (usually thymine dimers) resulting from UV irradiation
Single- or double-strand breaks
Breakage of phosphodiester bonds by ionizing radiation or chemical agents (e.g., bleomycin)
Cross-linked strands
Covalent linkage of two strands by bifunctional alkylating agents (e.g., mitomycin C)
3′-deoxyribose fragments
Disruption of deoxyribose structure by free radicals leading to strand breaks
Experimental demonstration of the proofreading function of E. coli DNA
polymerase I
Proofreading by DNA Polymerase Corrects Copying Errors
An artificial template [poly(dA)] and a corresponding primer end-labeled
with [3H]thymidine residues were constructed.
An “incorrect” cytidine labeled with 32P was then added to the 3′ end of
the primer. The template-primer complex was incubated with purified
DNA polymerase I.
In the presence of thymidine triphosphate (pppT), there was a rapid loss
of the [32P]cytidine and retention of all the [3 H]thymidine radioactivity.
This indicated that the enzyme removed only the terminal incorrect C and
then proceeded to add more T residues complementary to the template. In
the absence of pppT, however, both [3H]thymidine and [32P]cytidine were
lost, indicating that if the enzyme lacks pppT to polymerize, its 3′ → 5′
exonuclease activity will proceed to remove “correct” bases
Experimental demonstration of the proofreading function of E. coli DNA
polymerase I
Schematic model of the proofreading function of DNA polymerases
Chemical Carcinogens React with DNA Directly or after Activation
Direct-acting
carcinogens are
highly
electrophilic
compounds that
can react with
DNA.
Indirect-acting
carcinogens must
be metabolized
before they can
react with DNA.
All these
chemicals act as
mutagens.
DNA Damage Can Be Repaired by Several Mechanisms
Mismatch repair, which occurs immediately after DNA synthesis,
uses the parental strand as a template to correct an incorrect
nucleotide incorporated into the newly synthesized strand.
Excision repair entails removal of a damaged region by specialized
nuclease systems and then DNA synthesis to fill the gap.
Repair of double-strand DNA breaks in multicellular organisms occurs
primarily by an end-joining process.
DNA-repair mechanisms have been studied most extensively in E.
coli, using a combination of genetic and biochemical approaches.
The remarkably diverse collection of enzymatic repair mechanisms
revealed by these studies can be divided into three broad
categories:
Mismatch Repair of Single-Base Mispairs
Formation of a
spontaneous point
mutation by deamination of cytosine (C) to form uracil (U)
Model of mismatch repair by the E. coli MutHLS system
This repair system operates soon after
incorporation of a wrong base, before the
newly synthesized daughter strand
becomes methylate.
MutH binds specifically to a
hemimethylated GATC sequence, and
MutS binds to the site of a mismatch.
Binding of MutL protein simultaneously
to MutS and to a nearby MutH activates
the endonuclease activity of MutH, which
then cuts the unmethylated (daughter)
strand in the GATC sequence.
A stretch of the daughter strand
containing the mispaired base is excised,
followed by gap repair and ligation and
then methylation of the daughter strand.
Excision Repair
UV irradiation can cause adjacent
thymine residues in the same DNA
strand to become covalently
attached
The resulting thymine-thymine dimer
(cyclobutylthymine) may be repaired by
an excision-repair mechanism.
Excision repair of DNA by E. coli UvrABC mechanism
Repair of double-strand breaks by end-
joining of nonhomologous DNAs (dark and light blue),
that is, DNAs with dissimilar sequences
at their ends
End-Joining Repair of Nonhomologous
DNA
Inducible DNA-Repair Systems Are Error-Prone
• Both bacterial and eukaryotic cells have inducible DNA-repair
systems, which are expressed when DNA damage is so
extensive that replication may occur before constitutive
mechanisms can repair all the damage. The inducible SOS
repair system in bacteria is error-prone and thus generates and
perpetuates mutations.
• DNA-repair mechanisms that are ineffective or error-prone may
perpetuate mutations. This is a major way by which DNA
damage, caused by radiation or chemical carcinogens, induces
tumor formation. Thus, cellular DNA-repair processes have
been implicated both in protecting against and contributing to
the development of cancer.