DNA 的生物合成

1. DNA 的生物合成 Biosynthesis of DNA

2. The genetic central dogma

3. ⅠConcept of DNA replication or DNA biosynthesis • Semiconservative replication • The synthesis of a complementary DNA strand by forming phosphodiester linkages between nucleotides base-paired to template strand is catalyzed by large multienzyme complexes referred to as the DNA polymerases • The transmission of the genetic informations between DNA and DNA

4. parent DNA strand parent DNA molecules Daughter or new DNA strand daughter DNA molecules total conserve commingle semi conserve

5. The basic experiment condition • A experimental table • A centrifuge • A kind of bacterium • A few 14NH4Cl culture solutions • A few 15NH4Cl culture solutions • A few sucroses 7N14,15 / 1S22S22P3 Experimental basis of semiconservative replication

6. 密度梯度实验

7. Significance of semiconservative replication TGCA ACGT TGCA ACGT TGCA ACGT Genetic conservativeness

8. ⅡEnzymes in DNA replication • DNA polymerases • DNA helicase • DNA topoisomerase • DNA single strand binding protein • Primase • DNA ligase

9. proof read, repair, filling proof read, repair, filling veriest replicase substitute substitute major action major action in mitochondria DNA polymerases prokaryote polymeras I polymeras II polymeras III* eukaryote polymeras * polymeras  polymeras polymeras* polymeras 

10.           ’   ’         DNA polymerase III of E. coli

11. DNA polymerases function--1 5’3’ polymerase activity 5’ 3’ N N N + OH3' OH3' 5' P P P 5' P P  5’ 3’ N N N OH3' 5' P P P + PPi

12. DNA polymerases function--2 exonuclease activity * cut the fragment of primer and mutated fragments Polymerase I Polymerase II 5’ 5’ * Polymerase I Polymerase II Polymerase III Proof reading C A 3’ 3’ G G

13. high fidelity of DNA replication (1) The strict base complementary 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ G G A C T T A C

14. (2) the character of DNA polymerase selecting base 5’ T A A A T T A A T T A A T T T T The conformation of DNA polymerase III is changeable, as its affinity to nucleotide acids G 3’ C C G A A C G T C G C G C G G C C C G G T

15. (3) The proof reading function of DNA polymerase proof reading 5’ 5’ 5’ 5’ A C G 3’ 3’ 3’ 3’ T A A A A

16. DNA helicase Dna A, Dna B (rep), Dna C……Dna X Dna T Dna C Dna B Dna A Dna C Dna T

17. untwisting ligate cut DNA topoisomerase topoisomerase I: breaks single strand DNA, not requires ATP topoisomerase II: breaks double strands DNA, requires ATP

18. DNA topoisomerase normal helix helicase positive superhelix 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 DNA polimerase 1 2 3 4 5 6 7 8 9 10 11 topoisomerase negative superhelix

19. single stranded DNA-binding protein, SSB (1) SSB is consist of 177 amino acid residues in E.coli (2) tetrapolymer (3) a SSB= 32 nucleotides (4) Cooperative binding

20. The role of SSB Dna B SSB bank

21. Primase and Primosome  The primase is a RNA polymerase, which is different with that in the transcription.  The primase synthesizes the primers, which is used in DNA synthesis. The primer is a fragment of RNA  The length of primer is 10-20bp approximately  The helicases and other replicated factorshave binded with DNA, then the primasehave binds with them and formed a primosome at last.

22. the actions of primer and primase Parental DNA template 5’ 3’ SSB primase 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ DNA polymerase III 5’ 3’ 3’ 5’ New DNA strand

23. DNAligase 3’ 5’ P OH P OH 5’ 3’ ATP ADP+Pi 3’ 5’ 5’ 3’

24. The compare of several enzymes, which in formation of phosphodiester bond enzymes results provides 3’-OH provides 5’-P 1 DNA primer or dNTP (dNMP) n+1 polymerase extending DNA strand 2 DNA no continued two single strands no continue ligase  continue 3 DNA to break and put in order two put in order to DNA topoisomerase DNA single strands superhelical structure 4 primase extending primer NTP primer

25. Ⅲ Process of DNA replication • Initiation of DNA replication • Extension of DNA replication • Termination of DNA replication

26. Initiation of DNA replication Prokaryotic cell ( for instance E.coli) 1 A fixed origin (ori) 2 Bidirectional replication Eukaryotic cell a lot of origin

27. origin theta 1 2 O O O D D 3’ 21 2 1 21 21 3’ D Direction origin O O Eukaryotic cell Prokaryotic cell

28. leading strands Helicase, SSB ... Origin 2 1 lagging strands

29. The oriC of E coli. GATCTNTTTATTT---GATCTNTTNTATT---GATCTCTTATTAC 55 66 166 174201 209 237 245        1 13 17 29 32 44 TGTGGATTA---TTATACACA------TTTGGATAA---TTATCCACA palindrome structure DnaT DnaC DnaB DnaB DnaA DnaC DnaT

30. topoisomerase lagging strand 3’ polymerase III  subunit 5’ 3’ single strand binding protein 3’ 5’ polymerase III holoenzyme leading strand primer 5’ The extension of DNA replication

31. the termination of DNA replication origin 0% 82% 32% SV 40 E.coli 50% terminate point the terminate point of E.coli and SV40 virus

32. Parental DNA template strand New DNA strand the role of several enzyme in termination of DNA replication 3’ 5’ P OH 3’ 5’ RNA primer 3’ 5’ degradation P OH RNase 3’ 5’ Pol I filling 3’ 5’ P OH 3’ 5’ ligation Ligase 3’ 5’ 3’ 5’

33. Chemical reaction in DNA replication (dATP)m + (dCTP)n + (dGTP)y + (dGTP)z (dAMP + dCMP + dGMP + dGMP) m +n+ y+z + (m + n + y + z) PPi

34. helicase 5’ 3’ A C A G T G A T C A G A 5’ T G T C T C T 3’ primase primase H O P P P A G OH single strand binding protein O P PP HO O DNA polymerase Ⅲ ……dGTP/dTTP/dCTP/dTTP RNase DNA polymerase Ⅰ ligase O H 3’ 5’ single strand binding protein topoisomeraseⅡ