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

DNA replication steps overall

Answer 2

1. Licensing (G1)
2. Firing (S)
3. Progression (S)

Question 3

G1 phase: Pre-RC formation & origin licensing

Answer 3

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 4

S phase: Origin firing & initiation of replication

Answer 4

1. Expression of S phase cyclins (Clb5, Clb6) occurs at the G1-S transition: they phosphorylate sld3 & sld2
2. Phosphorylation of Sld2 & Sld3 increases their 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 5

The mitotic cyclin: Clb2/cdk1

Answer 5

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 6

Oncogene-induced replication stress

Answer 6

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 7

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

Answer 7

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
5. Rad53 also protects replication fork collapse by inhibiting Exo1, and it promotes dNTP synthesis through Dun1, which phosphorylates and inhibits Sml1, which inhibits ribonucleotide reductase, and Crt1, a transcriptional repressor of genes that encode ribonucleotide reductase

Question 8

Temporal activation of replication origins throughout S phase

Answer 8

1. Replication origins generally follow a temporal program of activation throughout S phase: the genome can be segmented into domains that replicate relatively synchronously at defined points during S phase
   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

Question 9

Nuclear organization

Answer 9

1. Late-replicating origins are often associated with silent chromatin that is highly compacted and transcriptionally inactive.
2. Early-replicating origins are often associated with highly transcribed open chromatin domains
3. Boundaries of the domains are marked by cohesion

Question 10

Replication timing in mammals

Answer 10

1. Timing of origin activation is regulated by several proteins – RIF1 delays activation of late origins by sequestering them from initiation factors

Question 11

What is MMS, HU, NZ and a triangle symbol

Answer 11

1. MMS: alkylating agent, methyl’s to nucleotides preventing DNA polymerase: DNA damaging agent
2. HU: inhibits RNR (DNA synthesis protein): decrease in nucleotides available, cells arrest in S phase
3. NZ: prevents progression in cell cycle by disrupting microtubules via depolymerization (arrest cell cycle), arrests cells in M phase
4. Triangle: gene deletion in yeast genetics