2. Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry Molecular Genetics and Clinical BiochemistryKSU DNA repair: mechanisms, methods to study DNA repair, syndromes

3. DNA Lesions That Require Repair

4. 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

5.  An artificial template [poly(dA)] and a corresponding primer end-labeled with [ 3 H]thymidine residues were constructed.  An “incorrect” cytidine labeled with 32 P 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 [ 32 P]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 [ 3 H]thymidine and [ 32 P]cytidine were lost, indicating that if the enzyme lacks pppT to polymerize, its 3′ → 5′ exonuclease activity will proceed to remove “correct” bases  An artificial template [poly(dA)] and a corresponding primer end-labeled with [ 3 H]thymidine residues were constructed.  An “incorrect” cytidine labeled with 32 P 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 [ 32 P]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 [ 3 H]thymidine and [ 32 P]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

6. 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.]

7. 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 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

8. 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 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.

9. 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

10.  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

11.  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

12. The identification and molecular cloning of the ras D 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

13. 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:

14. Mismatch Repair of Single-Base Mispairs Formation of a spontaneous point mutation by deamination of cytosine (C) to form uracil (U)

15. 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.]

16. Excision Repair

17. 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.]

18. 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.]

19. 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

20. 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

21. 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. 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.