Week 1: Replication Fork Stability

Question 1

How does the cell cycle push forward?

Answer 1

1. Cyclin-cdks activate the cyclin-cdks of the next cycle phase (they promote the expression of the next cyclin)
2. They do so via phosphorylation (these phosphorylation events change the substrates activity, stability or interactions with other proteins)

Question 2

Replication: G1 phase: Pre-RC formation & origin licensing

Answer 2

1. InS. cerevisiae, origins are Autonomously Replicating Sequences (ARS)
2. The ARS consensus sequence is recognized by the Origin of Replication Complex (ORC) – constitutively associated with ARS throughout the cell cycle.
3. ORC first recruits the ATPase Cdc6, the DNA replication licensing factor Cdt1 and then the Mcm2-7 helicase complex
4. Loading of the Mcm2-7 complex defines pre-RC formation; it confers DNA replication competence to the cell
5. at this stage Mcm2-7 helicase complex is associated with the ORC but it is still inactive!

Question 3

S phase: Origin firing & initiation of replication

Answer 3

1. Expression of S phase cyclins (Clb5, Clb6) occurs at the G1-S transition: they bind to cdk1 to phosphorylate sld3 & sld2
2. Phosphorylation of Sld2 & Sld3 increases sld2’s affinity for Dpb11 forming pre-loading complex …attaches to pre-RC
3. DDK (Dbf4-Cdc7) phosphorylates MCM2-7, increases its affinity for Cdc45 and GINS, leads to formation of the CMG complex (which represents the active helicase)

Question 4

The mitotic cyclin: Clb2/cdk1

Answer 4

1. phosphorylates ORC, prevents recruitment of Cdc6 & Cdt1
2. Phosphorylation of the Mcm2-7 complex may lead to nuclear export of the helicase complex, as well as the associated Cdt1
3. Phosphorylation of Cdc6 induces ubiquitin- dependent destruction by SCF (ubiquitin ligase) & proteasome, also leads to detach by the Clb2-Cdk1; Transcription of Cdc6 is also restricted to G1/S phase

Question 5

Oncogene-induced replication stress

Answer 5

1. Oncogene activation
   1a. Inefficient origin licensing=under replicated DNA
   1b. Re-replication=stalled/blocked replication forks
   1c. Premature origin activation=stalled/blocked replication forks
   1d. Impaired replication progression=stalled/blocked replication forks
   1e. ROS=DNA damage=Impaired replication progression =stalled/blocked replication forks
2. Stalled replication forks: via R loops, torsional stress, hairpin etc.

Question 6

General mechanism of the DNA replication checkpoint when DNA replication fork stalls due to a lesion

Answer 6

1. the MCM2-7 helicase may continue unwinding DNA
2. The resulting ssDNA is bound by RPA & may recruit and activate Mec1
3. Mec1, through the replication fork-associated adaptor protein P-Mrc1, activates P-Rad53
4. Rad53 then inhibits firing of late origins of replication through phosphorylation and inhibition of Sld3 and Dbf4

Question 7

Temporal activation of replication origins throughout S phase

Answer 7

1. Replication origins replicate at different times depending where they are: (timing is regulated via diff proteins such as RIF1 delaying late replicating origins to be late)
   1a. Early replicating origins: generally transcriptionally active gene-rich domains with specific epigenetic marks
   1b. Late replicating origins: generally low gene density, enriched in repressive epigenetic marks
2. These are separated via cohesin