telomerase Semiconservative replication Cell Division and DNA Replication

telomerase

הכפלה משומרת למחצה Semiconservative replication סרט ראשון

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

שאלה In man 104 to 105 sites

הליקאז Dna. B helicase loader Dna. C

g b NTP

DNA Polymerase Bacteria ►Single Ori להראות סרט שאלה ►Initiation or replication highly regulated ►Once initiated replication forks move at ~400 -500 bp/sec ►Replicate 4. 6 x 106 bp in ~40 minutes

DNA SYNTHESIS REACTION שאלה P CH 2 P Base O 5' end of strand CH 2 P Base O P CH 2 O Base CH 2 Base O H 20 + 3' P Synthesis reaction P 5' CH 2 3' OH P P OH P products O CH 2 O P Base OH 3' end of strand

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 bp – 400 mismatches – Probably all will be corrected by Pol III

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

DNA repair

topoisomerase

טופואיזומראז כמוטרפיה Etoposide – topo II inhibitor

DNA Replication DNA Polymerase held to DNA by clamp regulatory protein • • • Clamp protein releases DNA poly when runs into ds. DNA 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

Replication summery Replication Movie

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 forks and the completion of daughter chromosomes · Region 180 o from ori contains replication fork traps: ori Chromosome Ter sites

Replication Termination of the Bacterial Chromosome · One set of Ter sites arrest DNA forks progressing in the clockwise direction, a second set arrests forks in the counterclockwise direction: Chromosome Ter. B Ter. A

RNase H activity

תיאור הבעיה – קצוות חשופים של הכרומוזומים If this shoelace were a chromosome, then these two protective tips would be its

Tn. Am. Go 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 -14 kb long 8 -14 באדם אורכו בין telomere replication occurs late in the cell cycle ● עד 40 - מחלוקה אחת לשנייה מתקצרים הטלומארים ב . נוקלאוטידים 200

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

End-to-end fusion

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

T-loop seen in the electron microscope Green = Telomere-specific proteins

Telomerase Tel Conc

Telomerase is not active in most somatic cells

Cancer cells have telomerase

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.