רפליקציה טרנסקריפציהטרנסלציה Replication telomerase.
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Cell CycleRegulators
Replication Commitment
Cell Growth & Completion of
Replication
Cell Division
Cell Division and DNA Replication
Replication Initiation
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
origin of replicationכמה יש בגנום של הפרה?
. אחד1•
. אחד לכל כרומוזום2•
נוקלטוטידים100000. אחד כל כ-3•
נוקלאוטידים1000. אחד כל כ-4•
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
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
" המשלים – הגדיל תבנית י עפ לנוקלאוטיד נוקלאוטיד המוסיף אינזים פולימראז
DNA: דורש פולימראזקצה 1. עם OH' 3פרימאר.2TEMPLATE קריאה גדילנוקלאוטידים3.
תמיד מכיוון מפולימראז '3ל' 5סנטז
III
DNA PolymeraseBacteria
►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
מה תפקידו של הליקאז?
. לפתוח זיווגי בסיסים1•
. למנוע מהדנא לחזור למצב דו-גדילי2•
. ליצר פרימר של רנא3•
. לסנטז גדיל משלים4•
P
P
P
P
P
P
P
PP P
CH2
CH2
CH2
OH
OH
O
O
OBase
Base
Base
CH2
CH2
CH2
OH
O
O
OBase
Base
Base
5' end of strand
3' end of strand3'
5'
3'
H20+
Synthesis reaction
DNA SYNTHESIS REACTION
products
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
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
קריאה בדיקת
DNA פולימראז III הוא בעל ' 5' ל-3פעילות של אקסונוקלאז
וזה רק כאשר לא הוסיף את הנוקלאוטיד הבא
3
Polymerase III
5’ 3
’
Leading strand
base pairs
5’
5’
3’
3’
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:
Helicase +
Initiator Proteins
ATP
SSB Proteins
RNA Primer
primase
2Polymerase III
Lagging strand
Okazaki Fragments
1
RNA primer replaced by polymerase I& gap is sealed by ligase
Overall directionof replication
5’ 3’
5’
3’
5’
3’
3’ 5’
DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
Replication
Helicase: this unwinds DNA
DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
5’
5’
Overall directionof replication
5’
3’
5’
3’
3’
3’
DNA polymerase proofreads bases added and replaces incorrect nucleotides.
Replication
5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Leading strand synthesis continues in a 5’ to 3’ direction.
Replication
3’ 5’ 5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Okazaki fragment
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication
5’ 5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Okazaki fragment
Replication
5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication
3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication
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
DNA Synthesis
•Synthesis on leading and lagging strands
•Simultaneous replication occurs via looping of lagging strand
•Proofreading and error correction during DNA replication
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
Replication Termination of the Bacterial Chromosome
Termination: meeting of two replication forksand the completion of daughter chromosomes
Region 180o from ori contains replication fork traps:
ori
Ter sites
Chromosome
Replication Termination of the Bacterial Chromosome
TerATerB
One set of Ter sites arrest DNA forks progressing in the clockwise direction, asecond set arrests forks in the counterclockwise direction:
Chromosome
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
If this shoelace were a chromosome,
then these two protective tips would be its
תיאור הבעיה – קצוות חשופים של הכרומוזומים
CHROMOSOME
TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG
AATCCCAATCCC5’
3’
TELOMERE
פיתרון הבעיה – הוספת רצפים חוזרים לקצוות בסיום הרפליקציה
TnAmGo type of minisatellite repeat
TTAGGG – human
TTTAGGG – Arabidopsis
TTGGGG - Tetrahymena
TTAGG – Bombyx
TTTTAGGG – Chlamydomonas
TTTTGGGG – Oxytricha
TTAGGC - Ascaris
(TG)1-3 - Saccharomyces cereviceae
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• 8-14באדם אורכו בין • telomere replication occurs late in the cell
cycle• נוקלאוטידים בין 200 עד 40הטלומארים מתקצרים ב-
.חלוקה אחת לשניה
• 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
Functions
Telomerase
1. 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.
Dolly is aging too rapidly?….or was born 6 years oldDolly has developed pre-mature arthritis
מזה של כבש רגיל שנולד – ז"א נולדה 80%לדולי היה אורך טלומארים של שנים!6שהיא בת
נוקלאוטידים בכל חלוקת תא.200—50הטלומארים מתקצרים בקצב של
A Japanese-American Werner patient as a teenager (left), and at age 48 (Case #1 Epstein et al,1966, Medicine 45:177). She had eight children, two of whom were also affected. At 48, she hadhair loss and greying, thin extremities, chronic ulcerations of the ankles, atrophy of the skin and herthe right eye had been enucleated several years earlier due to acute glaucoma resulting from bilat-eral cateract extraction at the age of 27. She lived longer than many Werner patients, dying at 57.
Werner Patient
Teenager Age 48
Reverse Transcriptase
• Essential enzyme of RNA containing viruses such as HIV virus
• Synthesizes DNA in 5’ 3’ 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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