lecture 9

Question 1

describe initiation of replication in eukaryotes

Answer 1

chromosomes are linear and usually very long
- many origins per chromosome to replicate huge DNA content (not necessarily all activated)
- slower replication
- okazaki fragments are shorter
- histones wrap DNA forming nucleosome- 1st level of condensation
- their linear chromosomes have telomeres, causing a replication problem

Question 2

DNA replication takes place during __ phase of cel cycle in eukaryotes; formation of pre-replicative complex in __ phase

Answer 2

S
G1

Question 3

what is the origin of replication in yeast

Answer 3

ARS- 5 origins, not all activated

Question 4

replication forks at ARS is a __ step process, they are…

Answer 4

2
1- origin selection- formation of pre-replicative complex (aka assembly phase because formed at G1) - marks origins that will be used at replication
2- origin activation- DNA melting (2 strands) and enzymes expand replication bubble and formation of 2 replication forks (activity phase)

Question 5

is control of the cell cycle more regulated in prokaryotes or eukaryotes?

Answer 5

eukaryotes

Question 6

Origin selection is during __ phase and origin activation is during __ phase

Answer 6

G1
S

Question 7

describe origin selection (step 1)

Answer 7

eukaryotic initiator (proteins that bind origin), called ORC (heterohexamer)
- ORC is a trans-acting factor that binds to origin with ATP
- formation of pre-replication complex in G1 is a 3 step process:
1- binding of ORC initiator
2- binding of helicase loaders- Cdc6 & Cdt1
3- loading of helicases that encircle DNA duplex- Mcm2-7

Question 8

what are the components of the pre-RC

Answer 8

ORC, Cdc6, Cdt1, Mcm2-7

Question 9

the formation and activation of pre-RC is controlled by ___
explain

Answer 9

Cdk (cyclin dependent kinases)
- Cdk present low activity in G1 cycle (no phosphorylation for pre-RC formation), but needed for activation in step 2

Question 10

describe origin activation (step 2)

Answer 10

• helicase loaders (Cdc6 & Cdt1) dissociate
• Cdk and Ddk activate pre-RC
• once ORC is phosphorylated, dissociates from origin
• phosphorylation of several proteins leads to DNA melting and protein recruitment to start replication
• phosphorylated helicase now active
• phosphorylation by S-phase cdk’s necessary for replication fork assembly and confines initiation of replication to S phase

Question 11

describe cdk’s in step 2- origin activation

Answer 11

high cdk activity in S phase, activates complex and initiates replication; also inhibits new pre-RC from forming (only new initiation after whole cell cycle ends)

Question 12

new nucleosomes are assembled behind the replication fork…

Answer 12

histones are synthesized only during S phase- histones disassemble to allow replication machinery to move along DNA and are added as replication proceeds
- some histone parts are “inherited,” some are new- nucleosomes made of parental histones (ones already there) and new histones (duplicated DNA)
- spacing of histones every 200 nucleotides might be the reason for shorter okazaki fragments and slower replication