dr mohammad s alanazi, msc, phd molecular biology ksu dna repair: mechanisms, methods to study dna...

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.