2. Dr Mohammad S Alanazi, MSc, PhD Molecular Biology KSU 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 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

7. 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.

8. 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:

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

10. 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.

11. Excision Repair

12. Excision repair of DNA by E. coli UvrABC mechanism

13. 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

14. 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.