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Characteristics of replication

Characteristics of replication. Semi-conservative replication Bidirectional replication Semi-continuous replication. §1.1 Semi-Conservative Replication.

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Characteristics of replication

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  1. Characteristics of replication • Semi-conservative replication • Bidirectional replication • Semi-continuous replication

  2. §1.1 Semi-Conservative Replication • Conception: The original double-stranded DNA opens up and both strands serve as template for the synthesis of new DNA, The products of the reaction are two daughter double stranded DNA molecules. each of them has oneoriginal template strand and onenewly synthesized strand DNA.

  3. Bi-directional: replication is started in a single origin C site , extending in two direction , It process is called bi-directional replication. §1.2 Bidirectional Replication

  4. 3 3´ 5´ 3´ 3´ 5´ 5 Fork movedirection 3 5 Semi-continuous replication Leading strand Fork shift direction Lagging strand 5 Synthesis direction of leading strand accord with the replication fork shift direction, and synthesis direction of lagging strand is against the replication fork shift direction.

  5. DNA replication system 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

  6. Section 2Enzymology of DNA Replication

  7. The function of different DNA-pol of prokaryotes Polymerase Ⅰ: Single peptide chain, the function is proofreading and repairing . polymerase Ⅱ:Only has its activity without DNA-pol Ⅰand Ⅲ. polymerase Ⅲ:It is the most effective polymerase in synthesis of new strand DNA.

  8. All of DNA Polymerase possess the following biological activity. 1. 53 polymerizing(for replication) • Function: Recognizes the deoxynucleotide on the DNA template and then adds a complementary dNTP to the 3’-OH of the primer ,creating a 3’,5’phosphodiester bond on the daughter strand. 2. exonuclease (for proofreading) • Function: Check the base pair between parent and daughter strandstarting from the end of DNA,and remove mismatched deoxynucleotide from daughter strand.

  9. DNA-pol of eukaryotes DNA-pol : primase and extend the lagging strand DNA-pol : replication with low fidelity DNA-pol II DNA-pol : polymerization in mitochondria DNA-pol : elongation DNA-pol III DNA-pol : proofreading and filling gap DNA-pol I

  10. The initiation phase of DNA replication

  11. Unwinding of double helix DNA is required before replication, because the template must paired with its complement dNTP. Helicase can use the ATP to unwind double helix DNA forming a replication fork. §2.3 Helicase

  12. §2.4 SSB protein SSB(single-stranded DNA binding protein): When DNA was unwinded into two single strand by helicase, it intend to form double-stranded DNA again.SSB binds to single strand DNA and keep the single state of DNA in replication.

  13. During unwinding double helix DNA, the downstream double helix DNA over wrapped. The superhelix template needs to be released by topoisomerases. §2.5 Topoisomerase

  14. During replication , the synthesis of new strand may be discontinuous , to form many okazaki fragment . the DNA ligase can link two adjacent fragment by 3’-5’phosphodiester bond ,form the integrated daughter strand. §2.6 DNA Ligase

  15. Section 3DNA Replication Process

  16. Replication is a continuous process , to describe it clearly, we separate it into three stage : • Initiation: recognize the origine point, separate dsDNA, primer synthesis, … • Elongation: add dNTPs to the existing strand, form phosphodiester bonds, correct the mismatch bases, extending the DNA strand, … • Termination: stop the replication.

  17. §3.1 Replication of prokaryotes a. Initiation • The replication starts at a particular point called origin. • The origin of E. coliis 248 bp long and AT-rich.

  18. 58 66 166 174 201 209 237 245 1 13 17 29 32 44 GATTNTTTATTT ··· GATCTNTTNTATT ··· GATCTCTTATTAG ··· 3 5 5 3 Tandem Repeat Seq Inverted Repeat Seq ···TGTGGATTA-‖-TTATACACA-‖-TTTGGATAA-‖-TTATCCACA E.Coli oriC

  19. Primosome complex SSB

  20. Replication initiation related Pr.in procaryote protein Generic Name function Recognize ori C DnaA ( dnaA ) Unwinding Helicase DnaB ( dnaB ) DnaC ( dnaC ) DnaB cooperate DnaG ( dnaG ) Primase synthesis primer SSB stabilize ssDNA Topoisomerase unwind supercoiled DNA

  21. Releasing supercoil constraint • The supercoil constraints are generated ahead of the replication forks. • Topoisomerase binds to the dsDNA region just before the replication forks to release the supercoil constraint. • The negatively supercoiled DNA serves as a better template than the positively supercoiled DNA.

  22. b. Elongation dNTPs which is complementary to template are continuously connected to the primer or the nascent DNA chain by DNA-pol III. The nature of the chain elongation is the series formation of the phosphodiester bonds. 目 录

  23. Leading strand 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.

  24. Semi-continuous replication

  25. 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

  26. Proofreading and correction • 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.

  27. c. Termination • The replication of E. coli is bidirectional from one origin, and thetwo replication forksmust meet at one point called ter at 32. • All the primers will be removed, and all the fragments will be connected by DNA-pol I and ligase.

  28. Lagging strand synthesis • Primers on Okazaki fragments are digested by RNase. • The gaps are filled by DNA-pol I in the 5´→3´direction. • The nick between the 5´end of one fragment and the 3´end of the next fragment issealed by ligase.

  29. Genome of E. coli

  30. §3.2 Replication of Eukaryotes • DNA replication is closely related with cell cycle. • Multiple origins on one chromosome, and replications are activated simultaneously.

  31. Cell cycle

  32. Initiation • The eukaryotic origins are shorter than that of E. coli. • Requires DNA-pol  (primase activity) and DNA-pol  (polymerase activity). • Needs topoisomerase and replication factors (RF) to assist.

  33. b. Elongation • DNA replication and nucleosome assembling occur simultaneously. • Overall replication speed is compatible with that of prokaryotes.

  34. c. Termination

  35. Telomere • The terminal structure of eukaryotic DNA of chromosomes is called telomere. • Telomere is composed of terminal DNA sequence and protein. • The sequence of typical telomeres is rich in TTAGGG repeat sequence. • The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.

  36. Telomerase • The eukaryotic cells use telomerase to maintain the integrity of DNA telomere. • The telomerase is composed of telomerase RNA telomerase association protein telomerase reverse transcriptase • It is able to synthesize DNA using RNA as the template.

  37. Inchworm model

  38. Significance of Telomerase • Telomerase may play important roles in human aging and in cancer cell biology .

  39. aging Cell division Cell division telomerase Target for anti-cancer Immortalization

  40. Section 4Reverse Transcription

  41. §4.1 Reverse Transcription Reverse transcription: is a process in which genetic information is transmitted from RNA to DNA . It’s phenomenon often occure in the process that eukaryote cell be infected by RNA virus. In these RNA virus,the genetic information carrier is ssRNA instead of dsDNA (such as ssRNA viruses,HIV virus or tumour virus).

  42. §4.1 Reverse Transcription The genetic information carrier of some biological systems is ssRNA instead of dsDNA (such as ssRNA viruses). The information flow is from RNA to DNA, opposite to the normal process. This special replication mode is called reverse transcription.

  43. Viral infection of RNA virus During RNA virus infect Host cell,the genetic information must be transmitted from ssRNA to ds DNA ,and integrated into the chromosome of Host cell.Then replicate and transcribe with the DNA from Host cell.

  44. Reverse transcription Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA.

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