1. Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India

2. OC-SBT044-U01-01 Introduction Programmes and Courses  SEP – SBT044 – CP01  SEP - SBT044_CP101

3. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.3 Credits  Academic Inputs by Mrs. Rasika Bhore  M.sc (Microbiology) rasika.bhore@gmail.com

4. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of 40-60 minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about 40-60 minutes.  Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.  Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.  Appear several times, for all the Self-Tests, available for this course.  Student can use handouts for last minutes preparation just before end exam.

5. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to: Discuss various repair mechanisms used by cell to overcome defects introduced by mutation.

6. School of Science and Technology, Online Counseling Resource… Introduction  It is a challenging for a cell to carry out all functions normally & overcome the mutations.  For the survival, cell must have: The enzymatic machinery that replicates DNA must be inherently accurate. The cell must repair accidental damage to DNA that would destroy its function.  Cell possess various repairing mechanisms which maintain its stability. © 2007, YCMOU. All Rights Reserved.6

7. School of Science and Technology, Online Counseling Resource… Formation Of Dimer Photoproduct  UV irradiation promotes the formation of covalent bonds between adjacent pyrimidine residues creating Cyclobutane containing thymine dimer & mutagenic photoproduct.  As the COC bonds in this ring are shorter than the normal 0.34-nm base stacking in B-DNA, the DNA is distorted at this spot and is no longer a proper template for either replication or transcription. © 2007, YCMOU. All Rights Reserved.7

8. School of Science and Technology, Online Counseling Resource… DNA Repair Enzyme- Photolyase  Affected DNA can be repaired directly by some enzymes that simply reverse the chemical change.  Photolyase, a flavin and pterin dependent enzyme, binds at the dimer and uses the energy of visible light to break the cyclobutane ring, restoring the pyrimidines to their original form. This process called Photoreactivation. © 2007, YCMOU. All Rights Reserved.8

9. School of Science and Technology, Online Counseling Resource… O 6 -Methyl-guanine-methyltransferase  Damage by alkalyting chemicals like methyl- nitrosoguanidine causes methylation of base guanine at the oxygen of carbon atom 6.  The product o 6 -methylguanine often mispairs with thymine, changing GC to AT during replication.  O 6 -methyl-guanine methyltransferase, encoded by ada gene, recognizes o 6 -methylguanine in the DNA duplex & removes the methyl group by transferring it to an amino acid of the enzyme.  This enzyme also removes possibly disruptive methyl groups from phosphates of the DNA backbone. © 2007, YCMOU. All Rights Reserved.9

10. School of Science and Technology, Online Counseling Resource… Excision Repair System  Most important repair mechanism.  Damaged DNA is recognized, removed either as free bases or as nucleotides, and the gap is filled by synthesis of new DNA using the complementary strand as a template.  Types of excision repair: 1) Base excision repair 2) Nucleotide excision repair 3) Mismatch repair © 2007, YCMOU. All Rights Reserved.10

11. School of Science and Technology, Online Counseling Resource… Base Excision Repair  Base excision repair acts on single bases that have been damaged through oxidation or other chemical modification during normal cellular processes.  The damaged base is removed by DNA glycosylase, which cleaves the glycosidic bond, creating an AP (apurinic site or apyrimidinic site) site, where the sugar is with no base attached.  AP endonuclease that cleaves adjacent to AP site.  An exonuclease removes the deoxyribose- P and a number of additional residues.  The single base gap is filled by DNA polymerase and ligase. © 2007, YCMOU. All Rights Reserved.11

12. School of Science and Technology, Online Counseling Resource… Nucleotide Excision Repair  Nucleotide excision repair removes a whole oligonucleotide that contains the damage.  Thymine-thymine dimer residues are examples of damage caused by UV radiation.  In E. coli three genes involved (uvrA, B and C). The protein UvrA recognizes the damage, recruits UvrB and UvrC which cleave at 3' and 5' site of damage, respectively, producing an oligonucleotide of 12 or 13 bases.  Helicase necessary to remove oligonucleotide, DNA polymerase fills gap, ligase seals. © 2007, YCMOU. All Rights Reserved.12

13. School of Science and Technology, Online Counseling Resource… Genetic Defect  In mammalian cells, nucleotide excision repair is the main pathway for removal of carcinogenic lesions caused by sunlight or other mutagenic agents.  Such lesions are recognized by XPA protein whose defect leads to human skin disease xeroderma pigmentosum, an inherited human syndrome occurs when exposed to UV component of sunlight.  At sites recognized by XPA, a multiprotein endonuclease is assembled and the damaged strand is cleaved and repaired. © 2007, YCMOU. All Rights Reserved.13

14. School of Science and Technology, Online Counseling Resource… Mismatch Repair System  Recognizes mismatches resulting from replication.  Scans newly replicated DNA, identifies mismatch, excises the mismatched base specifically from new strand so error can be repaired.  The old strand of DNA be distinguished from the new strand after replication in E. coli because new strand not yet methylated at GATC sequences.  In E. coli mismatch repair is initiated by the protein MutS, which recognizes mismatch, and forms complex with MutL and MutH. Then MutH (an endonuclease) cleaves the unmethylated DNA strand at a GATC sequence.  Eukaryotes have a similar mismatch repair system, but the mechanism by which they identify the newly replicated DNA strand is not known. © 2007, YCMOU. All Rights Reserved.14

15. School of Science and Technology, Online Counseling Resource… Recombinational Repair  Recombinational repair make use of corresponding DNA segment from a separate but identical DNA molecule.  If both bases of a complementary pair are altered by mutagen, neither can act as a template for the other, which is overcome by this repair system.  The key to recombinational repair is an enzyme that anneals the sequences on either side of a lesion.  In E.coli, the RecA protein carries out this vital function. © 2007, YCMOU. All Rights Reserved.15

16. School of Science and Technology, Online Counseling Resource… SOS Repair  SOS genes are induced by severe damage which can even stop DNA synthesis, rather than merely changing the pairing properties of bases.  Example of an SOS- inducing lesion is pyrimidine dimer, which can not base pair at all.  When a replication fork meets a dimer, it stops & reinitiates some distance away, leaving a gap in the DNA to which the recombination RecA protein binds, induces SOS genes.  Some SOS genes are uvrA, uvrB, uvrC, & RecA itself all are induced after damage of DNA. © 2007, YCMOU. All Rights Reserved.16

17. School of Science and Technology, Online Counseling Resource… Error Prone Repair  Many times cell can continue DNA synthesis despite the apparently complete lack of template information at the site of a lesion.  A site which a apurinic or apyrimidinic i.e. lack the base is also repaired but usually makes a mutation in doing so, because there is no information to choose correct base to repair DNA molecule.  This is called ‘Error prone repair’.  E.coli genes umuC & umuD involves is error prone repair. © 2007, YCMOU. All Rights Reserved.17

18. School of Science and Technology, Online Counseling Resource… What We Learn………….  UV radiations are responsible for formation of cyclobutyl ring of thymine dimer, which can overcome by the enzyme photolyase.  O 6 -Methyl-guanine-methyltransferase removes methyl group from DNA introduced by chemical mutagens.  Base excision repair involves removal of damaged base creates a AP site which is filled by DNA polymerase & ligase.  uvrA, B and C genes acts in recognizing & removing whole damaged nucleotide.  In mismatch repair, proteins scans newly replicated DNA, identifies mismatch, excises the mismatched base specifically from new strand so error can be repaired.  Recombinational repair make use of corresponding DNA segment from a separate but identical DNA molecule.  SOS genes induced on severe damage to DNA, also involved in error prone repair.

19. School of Science and Technology, Online Counseling Resource… Critical Thinking Questions  While proofreading activity, DNA polymerase leaves about one mistake per 10 10-11 replicated base pairs. If a wrong nucleotide is introduced by the enzyme then it replicates during lifetime & intolerable mutation can occur. Which repair system can be useful to overcome this problem? © 2007, YCMOU. All Rights Reserved.19

20. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved. Tips For Critical Thinking Questions  Mismatch excision repair recognizes & removes wrong nucleotides.

21. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.21 Study Tips  Book Title: DNA in detail Author: J. D. Watson  Book Title: DNA repair mechanisms Author: Altman, Albert J.

22. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.22 Study Tips 1.www.web.virginia.eduwww.web.virginia.edu DNA repair 2. Howard-Flanders, P.1981. “ Inducible Repair Of DNA”.sci.Amer. 245:72-103

23. End of the Presentation Thank You !