Apricio - Lecture 6 Flashcards
(19 cards)
Replicon Model
Replicator: DNA sequence
Initiator:protein binds to replicator
Replicator (replication origin)
DNA sequence binds an “initiator” protein
* DNA synthesis starts at “the origin” which is usually within or adjacent to the replicator
Initiator
Protein; Binds to replicator
Regulates replication of the DNA
Unwinds (a little) DNA at the origin; recruits Helicase and other proteins for further replication
Eukaryotic Cell Cycle (overview)
- Major events (DNA replication - S phase, chromosome segregation - M phase) separated in time (cell cycle phases)
- Gap phases (G1 and G2): check points ensure all is well
- G1: decides to enter new cell cycle
- S: cell committed to complete the cycle
Pre-replicative Complex (at the replication origin)
- ORC - origin recognition complex binds replicator in Eukaryotic cells
- ORC recruits ==> Cdc6 and Cdt1 that recruits ==> MCM complex (Helicase)
- pre-RC assembled and activated once in each cell cycle
pre-RC activation and 2 replication Forks
pre-RC activation==>
- Helicase unwind DNA
- Recruit replication proteins (Ddk,Cdk,polymerases,cliding clamp, clamp loader…)
- assemble replication forks
Cdk: Cyclin-dependent kinase
Off (G1) = allow pre-RC assembly
On = cell begin new cell cycle; artivate pre-RC; breaks precursor of pre-RC (inhibit pre-RC assembly)
*Cdk ON until mitosis is complete and cell is in G1
* at the end of cell cycle, Cdk destroyed
Goal of replication
2 identical daughter cells
S phase
- Replication origins individually initiate replication; produce 2 forks per origin
- Cohesin proteins assemble holding two new DNA molecules together (cohesion)
- sister chromatid: fully replicated DNA molecules
cohesion
unless cohesion, newly synthesized chromosomes will be floating around.
Mitotic spindle senses tension
meiosis
homologous split 1st
sister chromatids 2nd
End Replication Problem
- DNA synthesis require a primer to initiate
- primer is later removed to complete DNA synthesis
- progressive shortening can occur if primer is located at the very end of the template (lagging strand)
=== Eukaryotic cell use telomerase to replicate the ends completely
Telomere
- ends of eukaryotic chromosomes
- simple 6 bp sequence
- repetitive
- chromosome caps; bind proteins (protect ends from exonucleolytic digestion or end-joining)
Telomerase extend 3’OH end
RNA molecule (has A,U,G,C)
- use RNA molecule as a template (reverese transcription); bind to DNA sequence using complementary RNA sequence
- extends 3’OH of DNA
- in eukaryotes, ends of linear DNA molecules; replicated by a unique mechanism; enzyme telomerase; synthesize short DNA repeats; elongate shortened telomere
Primase and DNA polymerase extend 5’ end
- Primase finds the starting point on extended ssDNA
- DNA polymerase extends from primer (creating new okazaki fragment)
- once okazaki fragmenet is repaired, telomere extension is finished (still has 3’ overhang)
Telomerase activity in Animals
- mice lacking telomerase less likely to form certain types of tumors
- telomerase and age?
- Inactive in most somatic cells == has replicative limit (Phase I, II, III - senescene)
- Active in germ line and stem cells
- Reactivated in immortalized (tumor) cells
- ALT (alternative lengthening of telomeres) occurs in some telomerase-negative cells/tumors
Telomere shortening is correlated with…
Aging In Vivo
Senescence In Vitro
Telomerase expression of human cells in culture
immortalizes human cells
no senescence stage observed for HEK cells with hTERT
ALT: T-Loop structures at telomeres
Alternative Lengthening of Telomeres - end of telomere anneals to another section of telomere
- -> elongated 3’ end of telomere invades other sites of the telomere creating “displacement loop”
- T-loops facilitate ALT in teomerase-negative cells
