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DNA repl.pptx
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- 2. INTRODUCTION âą Cells âą Fundamentalworking units of every living system. âą Every organism is composed of one of two radically different types of cells: âą prokaryotic cells âą eukaryotic cells which have DNA inside a nucleus. âą Prokaryotes and Eukaryotes are descended from primitive cells and the results of 3.5 billion years of evolution.
- 3. Cont------- âą All Cellshave common Cycles âą Born, eat, replicate, and die
- 5. Common features oforganisms âą Chemical energy is stored in ATP âą Genetic information is encoded by DNA âą Information is transcribed into RNA âą There is a common triplet genetic code âą some variations are known, however âą Translation into proteins involves ribosomes âą Shared metabolic pathways âą Similar proteins among diverse groups of organisms
- 6. All Life dependson 3 critical molecules 1.DNAs (Deoxyribonucleic acid) âą Hold information on how cell works 2.RNAs (Ribonucleic acid) âą Act to transfer short pieces of information to different parts of cell âą Provide templates to synthesize into protein 3.Proteins âą Form enzymes that send signals to other cells and regulate gene activity âą Form bodyâs major components
- 7. DNA, RNA, andthe Flow of Information The central dogma âą Formulated by Francis Crick âą Defines three major steps in the processing of genetic information âą 1. replication âą 2. Transcription âą 3. Translation
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- 9. DNA Replication âą Replicationis the duplication of DNA content before mitosis, i.e., the two DNA copies are divided equally between the new daughter cells. Each old strand will be copied into a complementary new strand. âą DNA replication is the process by which the genetic material is copied prior to distribution into daughter cells
- 10. Cont---- âą The originalDNA strands are used as templates for the synthesis of new strands âą It occurs very quickly, very accurately and at the appropriate time in the life cycle of the cell âą DNA replication relies on the complementarities of DNA strands âą The AT/GC rule.
- 11. Cont------- âą This processis called semi-conservative because it conserves only half of the original (parent) DNA molecule in the two daughter DNA molecules. One strand in each daughter molecule is completely new. âą which means that each daughter cell will receive a DNA strand from the mother cell and a complementary newly synthesized strand.
- 13. Cont---- Basic requirements forreplication A. Substrates âą The four deoxyribonucleosides triphosphate âą dATP âą dGTP âą dCTP âą dTTP
- 14. Cont---- âą B. Template âąFor DNA replication to occur the two chains have to unwind and separate. âą The separated strands serve as template for the synthesis of the new daughter strands. âą A template is required to direct the addition of the appropriate complimentary nucleotide to the newly synthesised DNA strand.
- 15. Cont---- âą C. Enzymes âąDNA synthesis is catalyzed by enzymes called DNA dependent DNA polymerase âą DNA polymerase I, is responsible for gap filling by nick translation of the RNA primers used during DNA replication into DNA sequence. âą DNA polymerase II, has proofreading and DNA repair function. âą DNA polymerase III, is the major enzyme responsible for DNA replication.
- 16. Cont---- DNA helicase âą Unwindsthe double helix into two single- stranded DNA. âą It breaks down hydrogen bonds between the bases at expense of ATP. Two ATP molecules are consumed for each base pair broken. Once separated, the single stranded DNA formed is stabilized by proteins called DNA-binding proteins.
- 17. Cont---- The single-stranded DNAbinding proteins (SSB) âą that prevent reannealing of DNA helix by binding the single stranded DNA created by helicase without interfering with DNA polymerase function.
- 18. Cont---- DNA gyrase âą Removespositive supercoils and introduces negative supercoils. Topoisomerases âą Catalyzes the relaxation of supercoiled DNA by breaking just one strand of DNA.
- 19. Primase âą DNA-dependent RNApolymerase that synthesizes short RNA primers (5-200 bases) with the help of DNA binding protein (primosome). âą RNA primer complementary and anti-parallel to the DNA is synthesized by primase. âą The DNA polymerase III requires a primer to elongate at the beginning of replication. The primer has a free 3'-OH to accept deoxy- ribonucleotides polymerized by DNA polymerase III.
- 20. Cont---- DNA ligase âą joinsthe free 3'-OH on C3 of one end to 5'-OH of phosphate on C5 of the other end of DNA.
- 21. STAGES OF REPLICATION Theprocess of replication can be divided in to three stages. âą Initiation âą Elongation âą Termination
- 22. Cont---- âą Initiation âą DNAreplication is initiated by the binding of DnaA proteins to the DnaA box sequences âą This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex. âą This causes the DNA to twist and puts torque on the nearby AT-rich region to denature and form a replication bubble
- 23. Cont---- âAT base pairsare held together by only 2 H bonds âCG base pairs are held together by 3 H bonds âTherefore, AT-rich regions of DNA denature more easily than CG-rich regions of DNA
- 24. Cont---- âą In thenext step, DnaB (also called helicase) binds to each strand of the separated double helix. Itâs job is to move along the DNA, progressively expand the replication fork in both directions. âą DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them
- 25. Cont---- âą This generatespositive supercoiling ahead of each replication fork so another enzyme, topoisomerase, travels ahead of the helicase and alleviates these supercoils âą Single-strand binding proteins (SSBPs) are also needed to bind to the separated DNA strands and keep them apart â Otherwise, the strands would simply reanneal
- 26. Cont---- âą After thehelicase, gyrase, and SSBPs are in place, short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase âą These short RNA strands start, or prime, DNA synthesis because DNA polymerase, the enzyme that copies DNA, cannot start a new strand on its own âą The RNA primers are later removed and replaced with DNA
- 27. Cont---- âą Elongation âą DNApolymerase III begins the process of elongation by adding free deoxy-ribonucleotides to the free 3'-OH end of the RNA primer in the 5'ï3' direction in a continuous manner (the leading strand) to the end of the DNA molecule or till reaching the flanking replication fork. âą This is done according to the base pairing rule: A- T and G-C using the 3'ï5' DNA strand as a template.
- 28. Cont---- âą The other5'ï3' the lagging strand of DNA, lags behind for a while until at least 1000 to 5000 nucleotides in prokaryotes (but only 150 - 250 nucleotides in eukaryotes) are exposed, where primase put a primer at the upper end of it at the replication fork, i.e., in 5'ï3' direction. âą DNA polymerase III covers the downstream(away from the replication fork) sequence by synthesizing at least 1000 DNA nucleotides.
- 29. Cont---- âą This processis repeated when a new enough stretch of DNA is again exposed due to the continuous progress of the leading strand, where, primase puts a new primer that is elongated by DNA polymerase III until it reaches the previous RNA primer, and so on. Each area enough for the DNA polymerase III to work on the lagging strand is called Okazaki fragment.
- 30. Cont---- âą Thus, onestrand is built towards the replication fork 5âï3â, i.e., the Leading strand, while the other strand is built in the opposite direction but also in 5'ï3', i.e., Lagging strands. âą Leading strand âą One RNA primer is made at the origin âą DNA pol III attaches nucleotides in a 5â to 3â direction as it slides toward the replication fork
- 31. Cont---- Lagging strand âą Synthesisis also in the 5â to 3â direction âHowever it occurs away from the replication fork âą Many RNA primers are required âą DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) âThese are termed Okazaki fragments after their discoverers
- 32. Cont---- âą Finally, theRNA primers are removed by the 5'ï3' nick translation by DNA polymerase I activity with a replacement into DNA nucleotides. âą The 5'-end of DNA fragments, built by DNA polymerase III, is linked to the 3'-end of the DNA fragment, built by the DNA polymerase I, by ATP-dependent ligase
- 33. Cont---- âą All DNApolymerases, whether bacterial or eukaryotic, share 2 very important limitations: âą 1. They cannot initiate DNA synthesis on their own. They require an RNA primer be laid down on the DNA first by DNA primase. âą 2. They can only âgrowâ a new DNA chain in the 5â to 3â direction.
- 34. Cont---- âą Proofreading âą DNAreplication exhibits a high degree of fidelity. âą Mistakes during the process are extremely rare âą There are several reasons why fidelity is high: âą 1. Instability of mismatched pairs â Complementary base pairs have much higher stability than mismatched pairs â This feature only accounts for part of the fidelity â It has an error rate of 1 per 1,000 nucleotides
- 35. Cont---- âą 2. Configurationof the DNA polymerase active site â DNA polymerase is unlikely to catalyze bond formation between mismatched pairs âą 3. Proofreading function of DNA polymerase â DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand â The enzyme uses its 3â to 5â exonuclease activity to remove the incorrect nucleotide
- 36. Cont---- âą C. Termination âąA specific protein, ter binding protein, binds termination sequences and prevents helicase from further unwinding of DNA and facilitates the termination of replication.
- 39. 5' 3' Primase RNA Primer DNApolymerase III Single-strand binding protein dATP dGTP dCTP dTTP ATP, GTP, CTP, TTP PPI Leading strand DNA polymerase I DNA ligase Helicase Lagging strand PPi 5' 5' 5' 3' 3' 5' Topoisomerases (Gyrase), Remove supercoiling