Gihan E-H Gawish, MSc, PhDAss. Professor

Molecular Genetics and Clinical Biochemistry

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.

[See A. Kornberg and T. A. Baker, 1992, DNA Replication, 2d ed., W. H. Freeman and Company.]

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

[Adapted from C. M. Joyce and T. T. Steitz 1995, J. Bacteriol. 177:6321; S. Bell and T. Baker, 1998, Cell 92:295.]

Genetic studies in E. coli have shown that proofreading does, indeed, play a critical role in maintaining sequence fidelity during replication.

Mutations in the gene encoding the ϵ subunit of DNA polymerase III inactivate the proofreading function and lead to a thousandfold increase in the rate of spontaneous mutations.

E. coli possesses an additional mechanism for checking the fidelity of DNA replication by identifying mispaired bases in newly replicated DNA.

This mismatch-repair machinery determines which strand is to be repaired by distinguishing the newly replicated strand (the one in which an error occurred during replication) from the template strand.

Experimental demonstration of the proofreading function of E. coli DNA

polymerase I Comment

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.

Direct-acting carcinogens

Indirect-acting carcinogens

Reactive electrophiles.

By chemically reacting with nitrogen and oxygen atoms in DNA

Modified distort the normal pattern of base pairing.

If not repaired, they would allow an incorrect nucleotide to be incorporated during replication.

Like chemical carcinogen, ethyl methanesulfonate (EMS), causes mutations.

Unreactive, water-insoluble compounds.

They can act as potent cancer inducers only after conversion to ultimate carcinogens by introduction of electrophilic centers.

Such metabolic activation of carcinogens is carried out by enzymes that are normal body constituents.

In animals, activation of indirect-acting carcinogens often is carried out by liver enzymes that detoxify noxious chemicals (e.g., therapeutic drugs, insecticides, polycyclic hydrocarbons, and some natural products).

Chemical Carcinogens React with DNA Directly or after Activation

Many of detoxify noxious chemicals are so fat-soluble that they would accumulate continually in fat cells and lipid membranes and not be excreted from the body.

The detoxification system works by converting such compounds to water-soluble derivatives, which the body can excrete.

Detoxification begins with a powerful series of oxidation reactions catalyzed by a set of proteins called cytochrome P-450.

These enzymes, which are bound to endoplasmic reticulum membranes, can oxidize even highly unreactive compounds such as polycyclic aromatic hydrocarbons.

Oxidation of polycyclic aromatics produces an epoxide, a very reactive electrophilic group:

Chemical Carcinogens React with DNA Directly or after Activation Indirect-acting

carcinogens

all chemical carcinogens act as mutagens.

The mutagenicity of most compounds identified as carcinogens for experimental animals has been demonstrated in simple bacterial assays.

Bacterial mutagenesis is the basis for routine tests for carcinogens.

Ames test, a chemical is incubated first with a liver extract to allow any metabolic activation to occur; it then is added to several different bacterial cultures designed to detect specific types of mutations.

A positive result in the Ames test shows that a compound has the potential to be carcinogenic, but does not indicate how potent it is.

The actual danger posed by any chemical is often assessed in animal studies, but even these are not a definitive indication of the danger to humans.

The Carcinogenic Effect of Chemicals Correlates with Their Mutagenicity

The identification and molecular cloning of the rasD oncogene

Addition of DNA from a human bladder carcinoma to a culture of mouse 3T3 cells causes about one cell in a million to divide abnormally and form a clone of transformed cells.

DNA from the initial focus of transformed mouse cells is isolated, and the oncogene is separated from adventitious human DNA by secondary transfer to mouse cells.

then cloned into bacteriophage λ; only the phage that receives human DNA hybridizes with an Alu probe.

The hybridizing phage should contain part or all of the transforming oncogene.

The strongest evidence that carcinogens act as mutagens comes from the observation that cellular DNA altered by exposure of cells to carcinogens

can change cultured cells into fast-growing cancer-type cells

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.

[Adapted from R. Kolodner, 1996, Genes and Develop. 10:1433; see also A. Sancar and J. Hearst, 1993, Science 259:1415.]

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 Two molecules of UvrA and one of UvrB form a complex that moves randomly along DNA

Once the complex encounters a lesion, conformational changes in DNA, powered by ATP hydrolysis, cause the helix to become locally denatured and kinked by 130°

After the UvrA dimer dissociates, the UvrC endonuclease binds and cuts the damaged strand at two sites separated by 12 or 13 bases

UvrB and UvrC then dissociate, and helicase II unwinds the damaged region, releasing the single-stranded fragment with the lesion, which is degraded to mononucleotides.

The gap is filled by DNA polymerase I, and the remaining nick is sealed by DNA ligase

[Adapted from A. Sancar and J. Hearst, 1993, Science 259:1415.]

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

[Adapted from G. Chu, 1997, J. Biol. Chem.

272:24097; M. Lieber et al., 1997, Curr. Opin. Genet.

Devel. 7:99.]

Eukaryotes Have DNA-Repair Systems Analogous to Those of E. Coli (mismatch repair)

Recent biochemical studies have shown that human cells carry out mismatch

repair by a process similar to that in E. Coli

The human MutL protein is recruited to the DNA by MutSα or MutSβ, but the

identity of the human nuclease (equivalent to MutH in E. coli) that actually cuts the

DNA is unknown.

Following cleavage, which can occur either 3′ or 5′ to the mismatch, an exonuclease

removes 100 – 200 nucleotides from the cut strand, spanning the mismatch.

DNA polymerase δ is principally responsible for filling in the gap, following which

the strands are sealed by the action of a DNA ligase

Eukaryotes Have DNA-Repair Systems Analogous to Those of E. Coli (excision-repair)

Genetic studies in eukaryotes ranging from

yeast to humans suggest that quite similar

excision-repair mechanisms are employed by

different organisms.

In higher eukaryotes, only a small fraction of

the genome is transcribed in any one cell

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.