רפליקציה

1. טרנסקריפציה טרנסלציה רפליקציה

2. Replication

3. telomerase

4. Mitosis animation פרופזה פרומטפזה מטפזה אנפזה טלופזה

6. Semiconservative replication הכפלה משומרת למחצה סרט ראשון

8. Cell Division and DNA Replication Cell Cycle Regulators Replication Initiation Replication Commitment Cell Growth & Completion of Replication Cell Division

9. In man 104 to 105 sites a replication rate of 2 kb/minute

10. Origins of replication • In E. coli only one site OriC • In man 104 to 105 sites • The direction of replication is bi-directional OriC OriC OriC

11. כמה origin of replicationיש בגנום של הפרה? • 1. אחד • 2. אחד לכל כרומוזום • 3. אחד כל כ-100000 נוקלטוטידים • 4. אחד כל כ-1000 נוקלאוטידים

12. מבנה אתר התחלת רפליקציה - ORI

13. הליקאז

14. DNA Replication DNA Helicase • Hydrolyze ATP when bound to ssDNA and opens up helix as it moves along DNA • Moves 1000 bp/sec • 2 helicases: one on leading and one on lagging strand • SSB proteins aid helicase by destabilizing unwound ss conformation

15. SSB proteins help DNA helicase destabilizing ssDNA • Binds to DNA with no sequence preference • Binds tighter to single strand than double • Keeps separated strands from rejoining

16. פרימאז פרימר: רצף קצר של נוקלאוטידים

17. 5’ GCATTCAGCAA 3’ 3’ AGUCG 5’ RNA ריבוז DNAדיאוקסי פרימר: רצף קצר של נוקלאוטידים

18. פולימראז – אינזים המוסיף נוקלאוטיד לנוקלאוטיד עפ"י תבנית הגדיל המשלים III פולימראז תמיד מסנטז מכיוון 5' ל3' • DNAפולימראז דורש: • פרימאר עם קצה 3' OH • TEMPLATEגדיל קריאה • נוקלאוטידים

19. g a b NTP

20. DNA Polymerase • Bacteria • Single Ori • Initiation or replication highly regulated • Once initiated replication forks move at ~400-500 bp/sec • Replicate 4.6 x 106 bp in ~40 minutes

21. מה תפקידו של הליקאז? • 1. לפתוח זיווגי בסיסים • 2. למנוע מהדנא לחזור למצב דו-גדילי • 3. ליצר פרימר של רנא • 4. לסנטז גדיל משלים

22. DNA SYNTHESIS REACTION 5' end of strand P P Base Base CH2 CH2 O O P P CH2 CH2 Base Base products O O H20 + 3' P P P P OH P Synthesis reaction Base CH2 P O CH2 5' Base O OH 3' 3' end of strand OH

23. DNA Pol III activity • 5’ to 3’ DNA polymerase • Very processive: Once it locks on it does not let go • Very active: Adds 1,000 nucleotides/sec! • High fidelity (מדויק): has a 3’ to 5’ exonuclease activity that removes mismatches

24. How good is Pol III? • 1 in 10,000 bases added are mismatched. • Of these, all but 1 in 1,000 are corrected by Pol III • E. coli genome 4,000,000 bp • 400 mismatches • Probably all will be corrected by Pol III

25. בדיקת קריאה DNA פולימראז III הוא בעל פעילות של 3' ל-5' אקסונוקלאז וזה רק כאשר לא הוסיף את הנוקלאוטיד הבא

26. בהפסקות

28. מזלג הרפליקציה

30. פרגמנט אוקזקי

32. ליגאז סרט רפליקציה

33. Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins: 5’ SSB Proteins Okazaki Fragments ATP 1 Polymerase III 2 Helicase + Initiator Proteins 3 Lagging strand 3’ primase base pairs 5’ Polymerase III RNA primer replaced by polymerase I & gap is sealed by ligase 5’  3’ Leading strand RNA Primer 3’

34. DNA repair

35. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Replication Helicase: this unwinds DNA DNA polymerase enzyme adds DNA nucleotides to the RNA primer.

36. Overall direction of replication 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides.

37. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Replication Leading strand synthesis continues in a 5’ to 3’ direction.

38. Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 5’ 3’ Replication Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

39. Replication Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

40. Replication 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

41. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Replication 3’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

42. Leading and Lagging strands

44. topoisomerase

45. טופואיזומראז כמוטרפיה Etoposide – topo II inhibitor

47. DNA Replication DNA Polymerase held to DNA by clamp regulatory protein • Clamp protein releases DNA poly when runs into dsDN • Forms ring around DNA helix • Assembly of clamp around DNA requires ATP hydrolysis • Remains on leading strand for long time; only on lagging strand for short time when it reaches 5’ end of proceeding Okazaki fragments

48. DNA Synthesis •Synthesis on leading and lagging strands •Proofreading and error correction during DNA replication •Simultaneous replication occurs via looping of lagging strand

49. Replication summery Replication Movie

50. Simultaneous Replication Occurs via Looping of the Lagging Strand •Helicase unwinds helix •SSBPs prevent closure •DNA gyrase reduces tension •Association of core polymerase with template •DNA synthesis •Not shown: pol I, ligase