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Chen Yonggang Zhejiang University Schools of Medicine

Biochemistry. Chen Yonggang Zhejiang University Schools of Medicine. Central dogma. replication transcription translation DNA RNA Protein. DNA Replication.

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Chen Yonggang Zhejiang University Schools of Medicine

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  1. Biochemistry Chen Yonggang Zhejiang University Schools of Medicine

  2. Central dogma replication transcription translation DNA RNA Protein

  3. DNA Replication .

  4. DNA Replication-Conservation of Information • DNA replication must be carried out every time a cell divides • Procaryotic growth involves cell division • Mitosis in eucaryotes involves cell division • DNA replication is template driven and synthesizes DNA in a semi-conservative manner dNTP + DNAnDNAn+1 +PPi

  5. DNA is replicated in a semi-conservative manner • Messelson & Stahl showed, using 15N-labelled DNA that the products of replication had intermediate density McKee 18.2

  6. Each separated DNA strand is duplicated to give two new double helices.

  7. DNA semiconservative replication Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules , each with one new strand and one old strand, this is semiconservative replication.

  8. The process which appeared simple initially is complex • Replication occurs just prior to cell division • The E coli chromosome is a single circular DNA double helix associated with proteins in a nucleoid • The E coli chromosome is negatively supercoiled and thus is quite compact and inaccessible McKee 17.16

  9. To allow the replication to occur supercoiling must be relaxed • A type I (single strand breaking) topoisomerase cleaves and relaxes the negative supercoiling ahead of the replication complex • The topoisomerase has a central hole through which the double helix passes. An intermediate is an enzyme-linked 3’OH • dnaA is displaced providing access for the next component needed for replication

  10. The first step in replication involves oriC • OriC is a 245 bp region, the origin of E coli replication • OriC contains 3 tandem repeats of a 13 bp sequence beginning in GATC and rich in AT bp • These repeats are weakly H-bonded and serve to provide 4 binding sites for a protein, dnaA, a start signal for replication • Replication proceeds in two directions - bidirectional

  11. dnaA allows binding of two other important proteins • dnaB is a DNA helicase which carries out the ATP- driven unwinding of the DNA double helix • dnaC is an important accessory protein which binds and is soon released • Together the three proteins utilize ATP to bend and separate the two strands of the bacterial chromosome

  12. SSB, a single strand binding tetramer stabilizes the initiation complex McKee 18.7

  13. DNA replication involves many enzymes • An RNA primase binds to the SSB stabilized melted helix • Since DNA polymerases require a primer and only extend that primer, the RNA primase (dnaG) in association with other proteins (primosome) synthesizes a 5-7 nucleotide primer using information from the template strand

  14. At the replication fork two strands are managed differently • The 5’ end of the primer(leading strand) is extended continuously by DNA polymerase III in a 5’→3’ direction by dNTPs • The primer on the lagging strand is also extended 5’→3’ by DNA polymerase III, in a discontinuous manner • Thus primer is made once on the leading strand and every 1000 nucleotides on the lagging strand

  15. The replication fork McKee 18.6

  16. DNA Polymerase III holoenzyme contains 10 distinct types of subunits • DNA Polymerase III is the primary replicase in E. coli • It has polymerase and 3’→5’ exonuclease activities • It functions as a dimer • It has great fidelity, only 1 error in 1010 bp • It is highly processive, sticking to the DNA for the entire trip through the chromosome • It has a rapid biosynthetic rate, synthesizing 1000 nt/sec

  17. The Pol III synthesizes DNA from dNTPs McKee 18.3

  18. The Dimer moves in one direction and synthesizes 5’→3’ • The leading strand is synthesized by addition of 5’-dNTPs in response to the template • The looped lagging strand is synthesized in Okazaki fragments using 5’-dNTPs • The lagging strand must be pieced together using DNA Polymerase I

  19. DNA polymerase III forms phosphodiester bonds • 2’-deoxynucleoside 5’ triphosphates are the activated intermediates needed for synthesis • Information from the parental strand provides the information for 5’→3’ synthesis (parental strand is read 3’→5’) • Thus each parental strand serves as the template for synthesis of a complementary strand

  20. Synthesis of a phosphodiester bond

  21. DNA Polymerase III is at the center of Replication McKee 18.8

  22. Top-down view of replication

  23. While DNA polymerase III does the replicating, DNAP I cleans up • Pol I(100kd) is a monomer of about 10% the size of Pol III(900kd) • It has three activities • It is a DNA polymerase • It is a 3’→5’ exonuclease • It is a 5’→3’ exonuclease • It is a processing and proofreading enzyme

  24. The three activities are on one polypeptide • The larger fragment(Klenow fragment) of 67kd contains the polymerase and the 3’→5’ exonuclease activity • The smaller, 36kd contains the 5’→3’ exonuclease activity

  25. As Pol III finishes, Pol I goes to work • The 5’→3’ exonuclease removes the RNA primer • The polymerase synthesizes DNA to fill the gap • Errors in Pol III synthesis are removed by 3’→5’ exonuclease • The function of DNA Pol II is not understood, although it apperas to be similar to Pol I

  26. Supercoiling was taken out by dnaB, DNA gyrase replaces it • Following synthesis of the strands, excision of RNA, replacement by DNA using Pol I, the supercoiling can be reinstated • DNA gyrase, an ATP- linked, energy requiring enzyme introduces negative supercoils to restore the original twist in the leading strand

  27. DNA ligase seals the Okazaki fragments and the completed double helical DNA • DNA polyI removes the primers and fills the gaps, DNA ligase seals the nicks and Okazaki fragments are connected • Pyrophosphate cleavage drives the reaction to completion • Termination occurs at a ter region and is mediated by a binding protein TBP • A type II(double stranded) topoisomerase is probably involved in helix dissociation(the two daughter DNA molecules separate)

  28. Enzymes and proteins involved in DNA replication 1, Topoisomerase 2, dnaA 1 : recognize the origin of replication dnaB(helixase):unwind double helix dnaC 3, SSB 4, Primase 5, DNA polymeraseIII, DNA polymeraseI 6, Ligase

  29. Happy Birthday • Samar and Barry

  30. Eucaryotic Replication is similar to that of procaryotes • Both have initiation, elongation and termination phases and are bidirectional • Both involve multiple DNA polymerases • Both involve multiple copies of the primary replicase which replicates strands differently • Replication rate is slower, but replication is rapid due to multiple replicons • Both require topoisomerases to unwind and rewind the DNA • Both require ligases

  31. Eucaryotic replication is distinct from that of procaryotes • There are 5 polymerases(α,β,γ,δ,ε) • The chromosomes are linear • There are multiple ori, and replication units • Replication only occurs during the S phase of the cell cycle • Telomeres restrict the number of times a replicon can be expressed

  32. Initiation of replication occurs at multiple ori • A large complex of proteins assembles at an ori (Origin Recognition Complex-ORC) • Details not for testing • A complex with helicase activity must bind and be activated • Replication Protein A (RPA ) binds and separates the strands(like SSB in E.coli) • RFC(replication factor C– a clamp loading factor) and PCNA(proliferating cell nuclear antigen) allows binding of Pol d to both the leading and lagging strand

  33. Binding of initiation factors to the lagging strand differs • Pol d is the main eucaryotic replication polymerase (Details not for testing) • Replication protein A(RPA) binds to the single strands • Pol a and a primase complex binds to the lagging strand • An RNA primer and 15-30 dNTPs are synthesized • Pol d binds and replicates one nucleosomes worth of Okazaki fragment

  34. Finishing and sealing of the lagging strand is different • Specific protein factors are important in finishing up replication (details not for testing) • DNA polymerase  remove the primers and DNA polymerase  excise errors • Topoisomerases induce supercoiling • DNA ligase seals the breaks • Chromosomes segregate • Replication bubbles merge • Telomeres determine the end of replication

  35. Eukaryotic DNA polymerases • Eukaryotes have at least 15 DNA Polymerases (5 most important): Pol α: acts as a primase (synthesizing a RNA primer), and then as a DNA Pol elongating that primer with DNA nucleotides. After a few hundred nucleotides elongation is taken over by Pol δ and ε. Pol β: is implicated in repairing DNA. Pol γ: replicates mitochondrial DNA. Pol δ: is the main polymerase in eukaryotes, it is highly processive and has 3'→5' exonuclease activity. Pol ε: may substitute for Pol δ in lagging strand synthesis, however the exact role is uncertain.

  36. Telomeres are GC rich self-complementary sequences at chromosome ends • Telomerase maintains the telomeres • Telomeres are repeat structures with a terminal loop • At each replication the telomeres are modified using an integral RNA template • Loss of telomeres limits replication • Cancer cells lose control of their telomeres

  37. Telomeres The structure at the ends of linear eukaryotic chromosomes, generally consist of many tandem copies of a short oligonucleotide sequence.

  38. Supercoiled DNA in Prokaryote

  39. Structure of Nucleosome

  40. Negative and positive supercoil

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