DNA Replication- General Concepts of DNA Replication.ppt
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The document provides a comprehensive overview of DNA replication, including its general concepts, characteristics, and the enzymatic processes involved. It explains semi-conservative, bidirectional, and semi-continuous replication mechanisms, highlighting the roles of various enzymes like DNA polymerases and topoisomerases. Additionally, it discusses replication fidelity and the mechanisms that correct errors during DNA synthesis.
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DNA Replication- General Concepts of DNA Replication.ppt
- 1. Section 1 General Concepts of DNA Replication
- 2. DNA replication • A reaction in which daughter DNAs are synthesized using the parental DNAs as the template. • Transferring the genetic information to the descendant generation with a high fidelity replication parental DNA daughter DNA 2
- 3. Daughter strand synthesis • Chemical formulation: (dNMP)n + dNTP (dNMP)n+1 + PPi DNA strand substrate elongated DNA strand • The nature of DNA replication is a series of 3´- 5´phosphodiester bond formation catalyzed by a group of enzymes. 3
- 5. Template: double stranded DNA Substrate: dNTP Primer: short RNA fragment with a free 3´-OH end Enzyme: DNA-dependent DNA polymerase (DDDP), other enzymes, protein factor DNA replication system 5
- 6. Characteristics of replication Semi-conservative replication Bidirectional replication Semi-continuous replication High fidelity 6
- 8. §1.2 Bidirectional Replication • Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand. • The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork. 8
- 10. Replication of prokaryotes The replication process starts from the origin, and proceeds in two opposite directions. It is named replication. 10
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- 13. Replication of eukaryotes • Chromosomes of eukaryotes have multiple origins. • The space between two adjacent origins is called the replicon, a functional unit of replication. 13
- 14. 5' 3' 3' 5' bidirectional replication fusion of bubbles 3' 5' 3' 5' 5' 3' 5' 3' origins of DNA replication (every ~150 kb) 14
- 15. §1.3 Semi-continuous Replication The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end. 15
- 16. 5' 3' 3' 5' 5' direction of unwinding 3' On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand. Leading strand 16
- 17. Semi-continuous replication 3' 5' 5' 3' replication direction Okazaki fragment 3' 5' leading strand 3' 5' 3' 5' replication fork 17
- 18. • Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 150-200 nt long for eukaryotes. • The daughter strand consisting of Okazaki fragments is called the lagging strand. Okazaki fragments 18
- 19. Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication. Semi-continuous replication 19
- 20. Section 2 Enzymology of DNA Replication
- 21. Enzymes and protein factors protein Mr # function Dna A protein 50,000 1 recognize origin Dna B protein 300,000 6 open dsDNA Dna C protein 29,000 1 assist Dna B binding DNA pol Elongate the DNA strands Dna G protein 60,000 1 synthesize RNA primer SSB 75,600 4 single-strand binding DNA topoisomerase 400,000 4 release supercoil constraint 21
- 22. • The first DNA- dependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in physiology or medicine in 1959. §2.1 DNA Polymerase DNA-pol of prokaryotes 22
- 23. • Later, DNA-pol II and DNA-pol III were identified in experiments using mutated E.coli cell line. • All of them possess the following biological activity. 1. 53 polymerizing 2. exonuclease 23
- 24. DNA-pol of E. coli 24
- 25. DNA-pol I • Mainly responsible for proofreading and filling the gaps, repairing DNA damage 25
- 26. Klenow fragment • small fragment (323 AA): having 5´→3´ exonuclease activity • large fragment (604 AA): called Klenow fragment, having DNA polymerization and 3´→5´exonuclease activity N end C end DNA-pol Ⅰ 26
- 27. DNA-pol II • Temporary functional when DNA-pol I and DNA-pol III are not functional • Still capable for doing synthesis on the damaged template • Participating in DNA repairing 27
- 28. DNA-pol III • A heterodimer enzyme composed of ten different subunits • Having the highest polymerization activity (105 nt/min) • The true enzyme responsible for the elongation process 28
- 29. Structure of DNA-pol III α： has 5´→ 3´ polymerizing activity ε：has 3´→ 5´ exonuclease activity and plays a key role to ensure the replication fidelity. θ: maintain heterodimer structure 29
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- 32. DNA-pol of eukaryotes DNA-pol : elongation DNA-pol III DNA-pol : initiate replication and synthesize primers DnaG, primase DNA-pol : replication with low fidelity DNA-pol : polymerization in mitochondria DNA-pol : proofreading and filling gap DNA-pol I repairing 32
- 33. §2.2 Primase • Also called DnaG • Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template. • Primers are short RNA fragments of a several decades of nucleotides long. 33
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- 35. • Primers provide free 3´-OH groups to react with the -P atom of dNTP to form phosphoester bonds. • Primase, DnaB, DnaC and an origin form a primosome complex at the initiation phase. 35
- 36. §2.3 Helicase • Also referred to as DnaB. • It opens the double strand DNA with consuming ATP. • The opening process with the assistance of DnaA and DnaC 36
- 37. §2.4 SSB protein • Stand for single strand DNA binding protein • SSB protein maintains the DNA template in the single strand form in order to • prevent the dsDNA formation; • protect the vulnerable ssDNA from nucleases. 37
- 38. §2.5 Topoisomerase • Opening the dsDNA will create supercoil ahead of replication forks. • The supercoil constraint needs to be released by topoisomerases. 38
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- 40. • The interconversion of topoisomers of dsDNA is catalyzed by a topoisomerase in a three-step process: • Cleavage of one or both strands of DNA • Passage of a segment of DNA through this break • Resealing of the DNA break 40
- 41. • Also called -protein in prokaryotes. • It cuts a phosphoester bond on one DNA strand, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut. Topoisomerase I (topo I) 41
- 42. • It is named gyrase in prokaryotes. • It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds. • It can change dsDNA into the negative supercoil state with consumption of ATP. Topoisomerase II (topo II) 42
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- 44. 3' 5' 5' 3' RNAase P OH 3' 5' 5' 3' DNA polymerase P 3' 5' 5' 3' dNTP DNAligase 3' 5' 5' 3' ATP §2.6 DNA Ligase 44
- 45. • Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand. • Sealing the nick in the process of replication, repairing, recombination, and splicing. 45
- 46. §2.7 Replication Fidelity • Replication based on the principle of base pairing is crucial to the high accuracy of the genetic information transfer. • Enzymes use two mechanisms to ensure the replication fidelity. – Proofreading and real-time correction – Base selection 46
- 47. • DNA-pol I has the function to correct the mismatched nucleotides. • It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication. Proofreading and correction 47
- 48. 3´→5´ exonuclease activity excise mismatched nuleotides 5´→3´ exonuclease activity cut primer or excise mutated segment C T T C A G G A G A A G T C C G G C G 5' 3' 3' 5' Exonuclease functions 48
- 49. Ensuring fidelity • Organism survival depends on accurate genome duplication – A mistake in DNA replication results in a permanent mutation in the genetic material & the possible elimination of that cell's progeny • 1. In E. coli, the chance of incorrect nucleotide incorporation is <10-9; fewer than 1 out of 1 billion nucleotides • 2. The E. coli genome contains ~4 x 106 nucleotide pairs, Thus, this error rate corresponds <1 nucleotide alteration for every 100 replication cycles DNA Replication… 49
- 50. • Ensuring fidelity Infidelity is detected based on the chemical conformation of nucleotide pairs – 1. DNA polymerase discriminates among 4 different precursors as they move in & out of active site – 2. Only one of them forms a proper geometric fit with the template, producing an A-T or G-C base pair that fits in the enzyme active site DNA Replication… 50
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- 53. • A large conformational change occurring on a millisecond timescale locks the correct nucleotide at the active site but promotes release of a mismatched nucleotide. • The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, whereas the same steps diminish the reaction probability of an incorrect substrate. 53
- 54. • Ensuring fidelity • If incoming nucleotide is correct, a conformational change occurs in which the polymerase rotates gripping the incoming nucleotide • If the newly formed base pair exhibits improper geometry, the active site cannot achieve the conformation required for catalysis & the incorrect nucleotide is not incorporated • In contrast, if the base pair exhibits proper geometry, the incoming nucleotide is covalently linked to the end of the growing strand • Despite this mechanism, incorrect pairings sometimes occur (~1 time for every 105 – 106 nucleotides) DNA Replication… 54
- 55. • Ensuring fidelity • Polymerase's 3'—>5' exonuclease activity takes care of mismatched bases • This can happen right after misincorporation or later on (mismatch repair) • Thus, there are three mechanisms to ensure accurate replication – 1. Accurate selection of nucleotides – 2. Immediate proofreading – 3. Postreplicative mismatch repair DNA Replication… 55