רפליקציה

טרנסקריפציה טרנסלציה

Replication

telomerase

Mitosis animation

פרופזהפרומטפזה

מטפזהאנפזה

טלופזה

Semiconservativereplication

למחצה הכפלה משומרת

סרט ראשון

Cell CycleRegulators

Replication Commitment

Cell Growth & Completion of

Replication

Cell Division

Cell Division and DNA Replication

Replication Initiation

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

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•

אתר מבנההתחלת

רפליקציה -ORI

הליקאז

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

פרימאז

פרימר: רצף קצר של נוקלאוטידים

5 ’GCATTCAGCAA 3’ 3 ’AGUCG 5’ RNA ריבוז

DNAדיאוקסיפרימר: רצף קצר של נוקלאוטידים

" 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

NTP

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

בהפסקות

הרפליקציה מזלג

אוקזקי פרגמנט

ליגאז

רפליקציה סרט

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

DNA repair

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

Leading and Lagging strands

topoisomerase

Etoposide – topo II inhibitor

כמוטרפיה

טופואיזומראז

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

Replication Movie

Replication summery

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

End-to-end fusion

Telomerase is composed of both RNA and protein

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.

and Primase

Telomeres are packaged into a unique structure -- a T-loop

Telomerase

Tel Conc

Telomerase is not active in most somatic cells

Cancer cells have telomerase

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.