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רפליקציה

טרנסקריפציה טרנסלציה

Replication

telomerase

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Mitosisחלוקת התא

DNAבתכלת כישור בירוק

בסגולצנטריול

Mitosis animation

פרופזהפרומטפזה

מטפזהאנפזהטלופזה

Eukaryotic Cell Cycle

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Phases of Mitosis

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Semiconservativereplication

משומרת למחצה

Semiconservativereplication

משומרת למחצההכפלה

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בפרוקריוטים רפליקציה

•In E. coli only one site OriC

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Cell CycleRegulators

Replication Commitment

Cell Growth & Completion of

Replication

Cell Division

Cell Division and DNA Replication

Replication Initiation

In man 104 to 105 sites

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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מבנה אתרהתחלת

רפליקציה-ORI

הליקאז

פרימאז

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5’ GCATTCAGCAA 3’3’ AGTCG 5’ RNA ריבוז

DNAדיאוקסי

י תבנית הגדיל המשלים"עפלנוקלאוטידנוקלאוטידהמוסיף אינזים–פולימראז

DNAדורשפולימראז:OH' 3עם קצה פרימאר1..2TEMPLATEגדיל קריאהנוקלאוטידים3.

'3ל' 5מכיוון יסנטזתמיד פולימראז

III

αβγ

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NTPsנוקלאוטידיםמוסיףפולימראזDNAכיצד

Nucleotide TriPosphare

DNA Polymerase

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

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

בדיקת קריאה

3’ to 5’

רק כאשר לא הוסיף את הבאהנוקלאוטיד

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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’ 5’ Exonuclease functions of Pol I and

Pol III – Use of RNA primers– DNA repair systems

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בהפסקות

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הרפליקציהמזלג

אוקזקיפרגמנט

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ליגאז

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Replication fork

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Leading and Lagging strands

topoisomerase

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Etoposide – topo II inhibitorכמוטרפיה

טופואיזומראז

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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Elongation in E. coli, part I

• 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

3’5’

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Elongation in E. coli, part II

• Pol III complex released from lagging strand template upon encountering previously synthesized Okazaki fragment

• Primosome synthesizes new primer

5’3’

Elongation in E. coli, part III

• 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

5’3’

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 catenatedDNA strands by a topoisomerase

oriC

TerGTerF

TerB TerC TerATerD

TerE

Clockwisefork trap

Counterclockwisefork trap

Counterclock-wise fork Clockwise

fork

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Replication Movie

Replication summery

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The Major DNA Polymerases

BACTERIAL

Enzyme Primary function

DNA Pol I (PolA) Major DNA repair enzymeDNA Pol II DNA repairDNA Pol III De novo synthesis of new DNA

_______________________________________________

MAMMALIAN

Enzyme Primary function Location

DNA Pol I (α) Strand synthesis initiation NucleusDNA Pol II (ε) DNA repair NucleusDNA Pol III (δ) Strand extension NucleusDNA Pol β DNA repair NucleusDNA Pol γ De novo synthesis of new DNA Mitochon.

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Eukaryotic DNA Polymerases• Replication of nuclear chromosomes involves

polymerase α and polymerase δ• Polymerase α (lagging strand replicase)

– Contains primase activity – Has no proofreading 3’ 5’ exonuclease activity

• Polymerase δ (leading strand replicase) – Lacks primase activity – Has 3’ 5’ exonuclease activity – Proliferating cell nuclear antigen

enhances processivity• Other polymerases (β, ε, γ) with

different roles also exist

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

DNA Polymerase III (Pol III)• Pol III is the principal E. coli

replication enzyme • Absence of Pol III is lethal • 5’ 3’ Exonuclease activity not

present • Composed of ten different kinds of

subunits that increase the activity and processivity of the core polymeraseο β subunit clamps around the

DNA and attaches to the core polymerase, greatly increasing the processivity of the enzyme

ο γ subunit opens the β 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’ 3’ and 3’ 5’ exonuclease activity

Exonuclease Activity of Pol I

• The 3’ 5’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 radiolabeledDNA

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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If this shoelace were a chromosome,

then these two protective tips would be its

קצוות חשופים של הכרומוזומים–תיאור הבעיה

CHROMOSOME

TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG

AATCCCAATCCC5’

3’

TELOMERE

הרפליקציה הוספת רצפים חוזרים לקצוות בסיום –פיתרון הבעיה

TTAGGG to plants

Ornithogalum umbellatum, Sykorova et al. 2003

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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

Rabl configuration

Telomere• senescent cells have shorter telomeres• קצריםטלומריםתאים מזדקנים בעלי • length differs between species• משתנה בין מינים שוניםהטלומראורך • in humans 8-14kb long• 8-14באדם אורכו בין • telomere replication occurs late in the cell

cycle• עד 40- בהטלומאריםבכל חלוקת תא הומאני מתקצרים

.נוקלאוטידים 200

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• Provide protection from enzymatic degradation and maintain chromosome stability

• ושומר על הכרומוזומיםאינזימטימונע פרוק • 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• של הכרומוזומיםהרפליקציהמאפשר את סיום

Functions

End-to-end fusion

טלומרים

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Template RNA

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

טלומראז

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טלומראז

Telomerase

pseudoknot

Template domain

Important for function

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How doth it worketh?

Commandeered from Brock Biology of Microorganisms, Ninth Ed.

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!

The telomere region before telomerase acts

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Telomerase binds to the template strand

The template strand is extended with repetitive DNA

Primase starts synthesis of the lagging strand

Primase

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The lagging strand is extended

DNA polymerase

Ligase seals the nick

Ligase

The RNA primer is removed

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Telomerase

ConcTel

בשמריםטלומר

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Telomerase is not active in most somatic cells

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Cancer cells have telomerase

Telomerase

Dolly is aging too rapidly?….or was born 6 years oldDolly has developed pre-mature arthritis

6א נולדה שהיא בת " ז– מזה של כבש רגיל שנולד 80% של טלומאריםלדולי היה אורך !שנים

. בכל חלוקת תאנוקלאוטידים 200—50 מתקצרים בקצב של הטלומארים

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Loss of telomeres in humans: loss of adult stem cell divisions

Loss of telomeres in humans: loss of adult stem cell divisions

Very similar to chemotherapy

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Telomerase, the Aging Process, and Cancer

• Inactive telomerase in Tetrahymena leads to shortened telomeres and eventual cell death

• In human germ-line cells, telomere lengths are maintained and telomerase is active

• Somatic cells lack telomerase and telomeres grow shorter with time

• In fibroblast cultures, a linear, inverse relationship exists between telomere length and the age of the donor

• Elimination of telomerase activity in somatic cells could protect multicellular organisms from cancer – Malignant cancer cells contain high levels of telomerase – Telomerase is being investigated as a target for cancer

chemotherapy

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• Expressed by both benign (שפיר) and malignant tumors

• 50% 0f the tumors have no detectable telomerase activity

• Expressed by >90% of neuroblastoma, skin, colon breast and uterine tumors

• Positivity less for brain, renal and hematologic neoplasms

Telomerse activity in various tumors

telomerase activity in tumors

High telomerase activity in tumors is due to– Reactivation of telomerase– Telomerase failed to switch off during

development – Tumor arises from a pre-existing

telomerase positive stem cell

A Japanese-American W erner 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 W erner patients, dying at 57.

Werner Patient

Teenager Age 48

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Reverse Transcriptase

• Essential enzyme of RNA containing viruses such as HIV virus

• Synthesizes DNA in 5’3’ 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.