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What is DNA and How is it Damaged and Repaired
- 1. Presented by - Amit Tiwari
- 2. What is DNA? • A polymeric molecule consisting of deoxyribonucleotide building blocks that in a double-stranded, double-helical form is the genetic material of all living organisms. • Each nucleotide consists of a pentose (five- carbon) sugar, a nitrogenous (nitrogen- containing) base (usually just called a base), and a phosphate group.
- 5. How DNA is damaged? DNA can be damaged in number of ways: • Exposure to UV light • Mechanical shearing • Phenol extraction • Dessication • Heating
- 7. DNA Repair • The process by which a cell uses a series of special enzymes to repair mutations (changes) in DNA and restore the DNA to its original state. • Maintaining the integrity of the information in DNA is a cellular imperative, supported by an elaborate set of DNA repair systems.
- 8. Types of DNA Repair • SOS Repair ( Error – prone repair system ) • Base Excision Repair • Nuclotide Excision Repair • Repair of Alkylation Damage
- 9. SOS RESPONSE • A system that repairs severely damaged bases in DNA by base excision and replacement, even if there is no template to guide base selection. • This process is a last resort for repair and is often the cause of mutations.
- 10. Historical Overview of SOS Response • Jean Weigle in 1953 observed reactivation of UV – irradiated lambda phage increased when irradiated phages were plated on previously irradiated Escherichia coli cells ( Weigle reactivation ) • Miroslav Radman concluded that in Escherichia coli there is DNA repair system dependent on LexA and RecA proteins and named it as “ SOS repair ’’
- 11. Recombination Proteins • recA and lexA genes were first to be recognized as being involved in SOS induction • 27 kDa LexA and 36 kDa RecA proteins were known as Recombination proteins operates in sexual life and genetic exchange of bacteria • RecA protein participates in genetic DNA exchange , in recF , recO , recR , recN and ruvABC- dependent recombinational DNA repair
- 12. Structure of LexA and RecA
- 13. SOS INDUCED DNA POLYMERASES • Five different DNA polymerases found in Escherichia coli : pol 1 to pol 5 • Pol I - fills gaps in course of DNA repair and in discontinuous DNA synthesis on lagging strand • Pol III - DNA polymerizing enzyme • Pol II - reactivates replicative DNA complex • Pol IV - induces of mutations in lambda phage and in episom F’ • Pol V - error prone translesion DNA polymerase
- 15. Mechanism of SOS Induction • In presence of ssDNA RecA protein interacts with LexA protein • LexA protein represses about 18 genes including itself • Each of those 18 genes has consensus sequence in their promoter called SOS box :- 5’-CTGX10CAG-3’ where X10 refers to any 10 bases
- 17. Mechanism of SOS Induction • LexA protein binds to SOS box and limits the trancription of genes • When RecA is activated it interacts with LexA to trigger autocatalytic properties of LexA
- 18. Mechanism of SOS Induction • RecA is not activated as there is no ssDNA , resulting in no longer destruction of LexA protein • LexA represses the suite of proteins involved in SOS response and SOS response is over.
- 20. Base Excision Repair • An enzyme-catalyzed process for repairing damaged DNA by removal of the altered base, followed by excision of the baseless nucleotide. • The correct nucleotide then is inserted in the gap.
- 21. Base Excision Repair • Major pathway for repair of modified bases , uracil misincorporation , oxidative damage • Base can be removed from nucleotide within DNA : by direct action of agents such as radiation , by spontaneous hydrolysis , by an attack of oxygen free radicals , or by DNA glycosylases.
- 22. DNA glycosylases • A family of DNA repair enzymes that recognize damaged nucleotide bases and remove them by hydrolyzing the N-glycosidic bond that attaches them to the sugar backbone of the DNA molecule.
- 23. Types of DNA glycosylases • Uracil DNA glycosylase • Helix - hairpin - helix glycosylase • 3-methyl-purine glycosylase (MPG) • Endonuclease VIII-like glycosylase
- 24. Uracil DNA glycosylase E. coli Uracil-DNA Glycosylase (UDG) catalyses the release of free uracil from uracil- containing DNA. UDG efficiently hydrolyzes uracil from single- stranded or double- stranded DNA, but not from oligomers (6 or fewer bases).
- 25. Helix – Hairpin – Helix glycosylase Structure of selected HhH Superfamily members HhH motif was first discovered in Endo III as a sequence – independent DNA binding motif.
- 26. 3-methyl-purine glycosylase (MPG) • Methylpurine-DNA glycosylase (MPG, or alkyladenine DNA glycosylase (AAG)) is a base excision-repair protein, catalyzing the first step in base excision repair by cleaving damaged DNA bases within double-stranded DNA to produce an abasic site. • MPG bends DNA by intercalating between the base pairs, causing the damaged base to flip out of the double helix and into the enzyme active site for cleavage.
- 27. 3-methyl-purine glycosylase (MPG) • It is responsible for the hydrolysis of the deoxyribose N-glycosidic bond, excising 3- methyladenine and 3-methylguanine from damaged DNA.
- 28. Endonuclease VIII-like glycosylase • Recognizes and repairs oxidized pyrimidines in the Base Excision Repair (BER) pathway.
- 29. AP endonuclease • Recognize and promote repair of AP sites generated either spontaneously or as the first step in BER. • These process the products of both monofunctional DNA glycosylases, which produce abasic sites, and the glycosylase/ AP lyases which cleave both base – to generate an abasic site.
- 30. AP endonuclease • It also cleaves the phosphodiester DNA backbone – to produce a 5’ phosphate and a 3’ αβ - unsaturated aldehyde group.
- 31. Mechanism of BER
- 32. Mechanism of BER
- 33. Mechanism of BER
- 34. Nucleotide Excision Repair • An enzyme-catalyzed process for removal of thymine dimers from DNA and synthesis of a new DNA segment complementary to the undamaged strand. • NER is important excision mechanism that removes DNA damage induced by UV.
- 35. Formation of DNA adducts • UV DNA damage results in bulky DNA adducts • These adducts are thymine dimers and 6,4- photoproducts
- 36. NER system proteins NER involves four proteins: • UvrA encoded by uvrA gene • UvrB encoded by uvrB gene • UvrC encoded by uvrC gene • UvrD encoded by uvrD gene
- 37. UvrA and UvrB protein structure
- 38. Mechanism of NER • UvrAB scans and finds DNA damage • UvrAs released; UvrC binds
- 39. Mechanism of NER • Cuts made 5’ and 3’ to damage • UvrD binds and unwinds region between cuts, releasing the damaged segment.
- 40. Mechanism of NER • DNA polymerase I fills in gap. • DNA ligase joins the DNA segments; repair is complete.
- 41. Repair of Alkylation Damage • Alkylating agents transfer alkyl groups usually methyl or ethyl group onto the bases at various locations , such as oxygen of carbon-6 in guanine. • In Escherichia coli , alkylation damage can be repaired by an enzyme called O6- Methylguanine-DNA methyltransferase, encoded by the ada gene.
- 42. O6-Methylguanine-DNA methyltransferase • O6-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O6-methylguanine. • It also repairs larger adducts on the O6- position of guanine, such as O(6)-[4-oxo-4-(3- pyridyl)butyl]guanine and O6- chloroethylguanine.
- 44. Repair of Alkylation Damage • O6-Methylguanine-DNA methyltransferase recognizes O6-Methylguanine in DNA and removes methyl group, thereby changing the base back to its original form. • Mutations of the genes encoding these repair enzymes result in a much higher rate of spontaneous mutation.
- 45. THANK YOU!