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טרנסקריפציה טרנסלציה רפליקציה
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Replication
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telomerase
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Mitosis חלוקת התא DNA בתכלת כישור בירוק צנטריול בסגול
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Mitosis animation פרופזה פרומטפזה מטפזה אנפזה טלופזה
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Eukaryotic Cell Cycle
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Phases of Mitosis
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Semiconservative replication משומרת למחצה
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Semiconservative replication הכפלה משומרת למחצה סרט ראשון
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Cell Division and DNA Replication
Cell Cycle Regulators Replication Initiation Replication Commitment Cell Growth & Completion of Replication Cell Division
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רפליקציה בפרוקריוטים In E. coli only one site OriC
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שאלה In man 104 to 105 sites
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Each eukaryotic chromosome is one linear DNA double helix
Average ~108 base pairs long With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days. Solution ---> DNA replication initiates at many different sites simultaneously. Rates are cell specific! Fig. 3.17
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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
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כמה origin of replication יש בגנום של הפרה?
1. אחד 2. אחד לכל כרומוזום 3. אחד כל כ נוקלאוטידים 4. אחד כל כ-1000 נוקלאוטידים
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מבנה אתר התחלת רפליקציה - ORI
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DnaBהליקאז helicase loader DnaC
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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
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Binds to DNA with no sequence preference
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
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פרימאז פרימר: רצף קצר של נוקלאוטידים
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5’ GCATTCAGCAA 3’ 3’ AGUCG 5’
RNA ריבוז DNAדיאוקסי פרימר: רצף קצר של נוקלאוטידים
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פולימראז – אינזים המוסיף נוקלאוטיד לנוקלאוטיד עפ"י תבנית הגדיל המשלים
III פולימראז תמיד מסנטז מכיוון 5' ל3' DNA פולימראז דורש: פרימאר עם קצה 3' OH TEMPLATE גדיל קריאה נוקלאוטידים
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g b a NTP
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כיצד DNA פולימראז מוסיף נוקלאוטידים
NTPs Nucleotide TriPosphare
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DNA Polymerase שאלה Bacteria Single Ori
להראות סרט Bacteria Single Ori Initiation or replication highly regulated Once initiated replication forks move at ~ bp/sec Replicate 4.6 x 106 bp in ~40 minutes שאלה
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מה תפקידו של הליקאז? 1. לפתוח זיווגי בסיסים
2. למנוע מהדנא לחזור למצב דו-גדילי 3. ליצר פרימר של רנא 4. לסנטז גדיל משלים
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DNA SYNTHESIS REACTION
שאלה 5' end of strand P P CH2 Base CH2 Base O O P P CH2 Base CH2 Base O O products H20 + P 3' P P P OH P Synthesis reaction Phosphodiester bonds CH2 Base P O 5' CH2 Base O OH 3' 3' end of strand OH
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איזה סוג קצה של דנא יהיה ב-5'?
5’ 3’ 1. קצה עם קבוצת OH. 2. קצה עם פוספאט אחד. 3. קצה עם שני פוספטים. 4. קצה עם שלושה פוספטים
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תבנית קריאה
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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
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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
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בדיקת קריאה DNA פולימראז III הוא בעל פעילות של 3' ל-5' אקסונוקלאז
וזה רק כאשר לא הוסיף את הנוקלאוטיד הבא
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Fidelity of Replication
Complexity of replication apparatus helps insure almost perfect fidelity of DNA replication Only 1 mispairing event occurs per every 108 to 1010 base pairs replicated in E. coli Accuracy due to: Balanced levels of dNTPs 3’ ’ Exonuclease functions of Pol I and Pol III Use of RNA primers DNA repair systems
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בהפסקות
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מזלג הרפליקציה
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פרגמנט אוקזקי
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ליגאז סרט רפליקציה
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Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:
base pairs 5’ 3’ Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins: SSB Proteins Polymerase III Lagging strand Okazaki Fragments 1 Helicase + Initiator Proteins ATP 2 3 RNA primer replaced by polymerase I & gap is sealed by ligase RNA Primer primase Polymerase III 5’ 3’ Leading strand
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DNA repair
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Replication Helicase: this unwinds DNA
Overall direction of replication 5’ 3’ Helicase: this unwinds DNA DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
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Replication DNA polymerase enzyme adds DNA nucleotides
5’ Overall direction of replication 3’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides.
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Replication Leading strand synthesis continues in a
5’ 3’ Overall direction of replication Leading strand synthesis continues in a 5’ to 3’ direction.
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Replication Leading strand synthesis continues in a
3’ 5’ Overall direction of replication Okazaki fragment Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
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Replication Leading strand synthesis continues in a
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.
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Replication Leading strand synthesis continues in a
3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
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Replication Leading strand synthesis continues in a
5’ 3’ 3’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
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Replication fork
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לפניך גדיל של DNA שעובר רפליקציה. איזה גדיל משלים יסונטאז?
5’ 3’ ב א 1. מ-ב יסונטאז הגדיל הנגרר ומ-א הגדיל המוביל. 2. מ-ב יסונטאז הגדיל המוביל ומ-א הגדיל הנגרר. 3. מ-שניהם הגדיל המוביל. 4. משניהם הגדיל הנגרר.
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topoisomerase
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טופואיזומראז כמוטרפיה Etoposide – topo II inhibitor
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How are the synthesis of the leading and lagging strands coordinated?
Most of the details are not clear but the problem is daunting Priming the lagging strand Synthesizing, releasing and picking up the lagging strand And all this has to be done while adding 1,000 bp/sec
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Leading and Lagging strands
להתכונן לשאלה
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DNA Replication DNA Polymerase held to DNA by clamp regulatory protein
Clamp protein releases DNA poly when runs into dsDNA 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
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Elongation in E. coli, part I
3’ 5’ Leading strand synthesis is continuous Primosome intermittently primes lagging strand synthesis Lagging strand template looped to permit simultaneous replication of the leading and lagging strands Collision looming with previous Okazaki fragment
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Elongation in E. coli, part II
5’ 3’ Pol III complex released from lagging strand template upon encountering previously synthesized Okazaki fragment Primosome synthesizes new primer
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Elongation in E. coli, part III
5’ 3’ Pol III rebinds the lagging strand template RNA primer is extended to form a new Okazaki fragment Leading strand synthesis is always ahead of lagging strand synthesis Result of this sequence is a series of RNA-primed fragments separated by nicks on the lagging strand
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Termination Ter (terminus) sites create a trap that replication forks cannot exit Ter sequences bind the Tus protein, which inhibits DnaB helicase Tus-Ter complex arrests a replication fork from only one direction – prevents overreplication by one replication fork Final step of DNA replication in E. coli is the unlinking of catenated DNA strands by a topoisomerase
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DNA Synthesis •Synthesis on leading and lagging strands
•Proofreading and error correction during DNA replication •Simultaneous replication occurs via looping of lagging strand
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Replication summery Replication Movie
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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
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Replication Termination of the
Bacterial Chromosome Termination: meeting of two replication forks and the completion of daughter chromosomes Region 180o from ori contains replication fork traps: ori Ter sites Chromosome
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Replication Termination of the
Bacterial Chromosome TerA TerB One set of Ter sites arrest DNA forks progressing in the clockwise direction, a second set arrests forks in the counterclockwise direction: Chromosome
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The Major DNA Polymerases
BACTERIAL Enzyme Primary function DNA Pol I (PolA) Major DNA repair enzyme DNA Pol II DNA repair DNA Pol III De novo synthesis of new DNA _______________________________________________ MAMMALIAN Enzyme Primary function Location DNA Pol I () Strand synthesis initiation Nucleus DNA Pol II () DNA repair Nucleus DNA Pol III () Strand extension Nucleus DNA Pol DNA repair Nucleus DNA Pol De novo synthesis of new DNA Mitochon.
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Eukaryotic DNA Polymerases
Enzyme Location Function Pol (alpha) Nucleus DNA replication includes RNA primase activity, starts DNA strand Pol (gamma) Nucleus DNA replication replaces Pol to extend DNA strand, proofreads Pol (epsilon) Nucleus DNA replication similar to Pol , shown to be required by yeast mutants Pol (beta) Nucleus DNA repair Pol (zeta) Nucleus DNA repair Pol (gamma) Mitochondria DNA replication
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RNaeH activity
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Eukaryotic DNA Polymerases
Replication of nuclear chromosomes involves polymerase a and polymerase d Polymerase a (lagging strand replicase) Contains primase activity Has no proofreading 3’ ’ exonuclease activity Polymerase d (leading strand replicase) Lacks primase activity Has 3’ ’ exonuclease activity Proliferating cell nuclear antigen enhances processivity Other polymerases (b, e, g) with different roles also exist
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Elongation DnaB helicase unwinds double helix ahead of the advancing replication fork and SSB protein prevents re-annealing of single stranded regions RNA primer is synthesized at the replication fork by primase for the leading strand, then for the lagging strand Two Pol III complexes carry out DNA elongation, one for the leading strand and one for the lagging strand The lagging strand template is looped so that the replisome moves as a unit in the 5’ 3’ direction
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DNA Polymerase III (Pol III)
Pol III is the principal E. coli replication enzyme Absence of Pol III is lethal 5’ ’ Exonuclease activity not present Composed of ten different kinds of subunits that increase the activity and processivity of the core polymerase b subunit clamps around the DNA and attaches to the core polymerase, greatly increasing the processivity of the enzyme g subunit opens the b clamp to load and unload the core polymerase onto the DNA template
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DNA Polymerase I (Pol I)
First DNA polymerase discovered (Kornberg, 1957) Isolated based on its ability to incorporate [14C]thymidine into DNA Consists of a single 928 amino acid polypeptide Possesses 5’ ’ and 3’ ’ exonuclease activity
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Exonuclease Activity of Pol I
The 3’ ’ exonuclease activity serves a proofreading function Pol I’s most important function is not replication, but replacement of RNA primers in lagging strand synthesis Pol I is used to repair damaged DNA and is also used in the lab to prepare radiolabeled DNA
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DNA Ligase RNA primers removed and gaps filled by Pol I
DNA ligase seals nicks between a 3’-OH and a 5’-phosphate Ligase from E. coli requires NAD+, eukaryotic ligases require ATP Reaction mechanism involves a phosphoamide intermediate between the ligase and the adenyl group
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Telomeres תיאור הבעיה – קצוות חשופים של הכרומוזומים
If this shoelace were a chromosome, then these two protective tips would be its Telomeres
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CHROMOSOME פיתרון הבעיה – הוספת רצפים חוזרים לקצוות בסיום הרפליקציה
TELOMERE 3’ TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 5’ AATCCCAATCCC
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TnAmGo type of minisatellite repeat
TTAGGG – human TTTAGGG – Arabidopsis TTGGGG - Tetrahymena TTAGG – Bombyx TTTTAGGG – Chlamydomonas TTTTGGGG – Oxytricha TTAGGC - Ascaris (TG)1-3 - Saccharomyces cereviceae
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Rabl configuration
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Telomere senescent cells have shorter telomeres
תאים מזדקנים בעלי טלומרים קצרים length differs between species אורך הטלומר משתנה בין מינים שונים in humans 8-14kb long באדם אורכו בין 8-14 telomere replication occurs late in the cell cycle בכל חלוקת תא הומאני מתקצרים הטלומארים ב-40 עד 200 נוקלאוטידים.
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Functions Provide protection from enzymatic degradation and maintain chromosome stability מונע פרוק אינזימטי ושומר על הכרומוזומים Organization of the cellular nucleus by serving as attaching points to the nuclear matrix משמש נקודות מעגן למערך רשת הגרעין Allows end of linear DNA to be replicated completely מאפשר את סיום הרפליקציה של הכרומוזומים
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End-to-end fusion
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טלומרים
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Template RNA
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Telomeres and Telomerase
DNA polymerases cannot replicate the extreme 5’-ends of chromosomes due to RNA priming (primer gap) Telomerase maintains the length of chromosome ends (telomeres) RNA-dependent DNA polymerase Contains an RNA component that acts as a template for nucleotide addition
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טלומראז
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Telomerase is composed of both RNA and protein
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Telomerase Telomerase binds to the telomer and the internal RNA component aligns with the existing telomer repeats. 2. Telomerase synthesizes new repeats using its own RNA component as a template 3. Telomerase repositions itself on the chromosome and the RNA template hybridizes with the DNA once more.
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and Primase
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טלומראז
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Telomeres are packaged into a unique structure -- a T-loop
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T-loop seen in the electron microscope Green = Telomere-specific proteins
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כמה חזרות (בערך) יש בכול טלומאר?
אחת 166 1666 16666
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Telomerase Template domain Important for function pseudoknot
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How doth it worketh? 1)RNA portion of enzyme recognizes telomeric repeats 2)Binds and begins adding repeats to overhanging 3’end 3)Eventually the overhang is long enough and primase can work its magic and there we have a functional lagging strand Okazaki fragment. 4)Hurrah! Commandeered from Brock Biology of Microorganisms, Ninth Ed.
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The telomere region before telomerase acts
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Telomerase binds to the template strand
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The template strand is extended with repetitive DNA
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Primase starts synthesis of the lagging strand
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The lagging strand is extended
DNA polymerase
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Ligase seals the nick Ligase
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The RNA primer is removed
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Telomerase Tel Conc
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Telomerase is not active in most somatic cells
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Cancer cells have telomerase
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לדולי היה אורך טלומארים של 80% מזה של כבש רגיל שנולד – ז"א נולדה שהיא בת 6 שנים!
הטלומארים מתקצרים בקצב של 50—200 נוקלאוטידים בכל חלוקת תא. Dolly is aging too rapidly?….or was born 6 years old Dolly has developed pre-mature arthritis
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Werner Patient Teenager Age 48
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Reverse Transcriptase
Essential enzyme of RNA containing viruses such as HIV virus Synthesizes DNA in 5’ ’ direction from an RNA template Viral RNA is degraded by RNase H domain of the protein, then complementary DNA is synthesized DNA integrated into host cell chromosome from Molecular Biology of the Cell, Alberts, Bray, Lewis, Raff, Roberts, and Watson, Garland Publishing, New York 1994.
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