Apricio - Lecture 6

Question 1

Replicon Model

Answer 1

Replicator: DNA sequence
Initiator:protein binds to replicator

Question 2

Replicator (replication origin)

Answer 2

DNA sequence binds an “initiator” protein

* DNA synthesis starts at “the origin” which is usually within or adjacent to the replicator

Question 3

Initiator

Answer 3

Protein; Binds to replicator
Regulates replication of the DNA
Unwinds (a little) DNA at the origin; recruits Helicase and other proteins for further replication

Question 4

Eukaryotic Cell Cycle (overview)

Answer 4

1. Major events (DNA replication - S phase, chromosome segregation - M phase) separated in time (cell cycle phases)
2. Gap phases (G1 and G2): check points ensure all is well
3. G1: decides to enter new cell cycle
4. S: cell committed to complete the cycle

Question 5

Pre-replicative Complex (at the replication origin)

Answer 5

1. ORC - origin recognition complex binds replicator in Eukaryotic cells
2. ORC recruits ==> Cdc6 and Cdt1 that recruits ==> MCM complex (Helicase)
3. pre-RC assembled and activated once in each cell cycle

Question 6

pre-RC activation and 2 replication Forks

Answer 6

pre-RC activation==>

1. Helicase unwind DNA
2. Recruit replication proteins (Ddk,Cdk,polymerases,cliding clamp, clamp loader…)
3. assemble replication forks

Question 7

Cdk: Cyclin-dependent kinase

Answer 7

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

Question 8

Goal of replication

Answer 8

2 identical daughter cells

Question 9

S phase

Answer 9

1. Replication origins individually initiate replication; produce 2 forks per origin
2. Cohesin proteins assemble holding two new DNA molecules together (cohesion)
3. sister chromatid: fully replicated DNA molecules

Question 10

cohesion

Answer 10

unless cohesion, newly synthesized chromosomes will be floating around.
Mitotic spindle senses tension

Question 11

meiosis

Answer 11

homologous split 1st

sister chromatids 2nd

Question 12

End Replication Problem

Answer 12

• 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

Question 13

Telomere

Answer 13

• ends of eukaryotic chromosomes
• simple 6 bp sequence
• repetitive
• chromosome caps; bind proteins (protect ends from exonucleolytic digestion or end-joining)

Question 14

Telomerase extend 3’OH end

Answer 14

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

Question 15

Primase and DNA polymerase extend 5’ end

Answer 15

1. Primase finds the starting point on extended ssDNA
2. DNA polymerase extends from primer (creating new okazaki fragment)
3. once okazaki fragmenet is repaired, telomere extension is finished (still has 3’ overhang)

Question 16

Telomerase activity in Animals

• mice lacking telomerase less likely to form certain types of tumors
• telomerase and age?

Answer 16

1. Inactive in most somatic cells == has replicative limit (Phase I, II, III - senescene)
2. Active in germ line and stem cells
3. Reactivated in immortalized (tumor) cells
4. ALT (alternative lengthening of telomeres) occurs in some telomerase-negative cells/tumors

Question 17

Telomere shortening is correlated with…

Answer 17

Aging In Vivo

Senescence In Vitro

Question 18

Telomerase expression of human cells in culture

Answer 18

immortalizes human cells

no senescence stage observed for HEK cells with hTERT

Question 19

ALT: T-Loop structures at telomeres

Answer 19

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