Week 2: Damage-induced Phosphorylation Of SLD3 Is Important To Block Late Origin Firing

Question 1

What happens when sld3 and dbf4 is phosphorylated

Answer 1

1. Phosphorylation of sld3 (via Clb5/6) and activation of dbf4 allow for replication to occur
2. HOWEVER, when sld3 and dbf4 are phosphorylated/inhibited via Rad53 it blocks late origin firing so that cells proceed through S phase faster through inappropriate late origin firing (in DNA damage)

Question 2

What is the importance of sld3

Answer 2

1. Required for the initiation of DNA replication in budding yeast (binds to dbp11->forms Pre loading complex)
2. In dna damage, it needs to be turned off to block late origin firing which depends on Mec1(ATM) and Rad53 (Chk2)

Question 3

What question were authors trying to address

Answer 3

1. What is the Rad53 substrate whose phosphorylation inhibits activation of late replication origins in response to DNA damage
2. Main finding of paper: sld3 is phosphorylated via rad53 which blocks late origin firing in DNA damage

Question 4

Figure 1A

Answer 4

1. Objective: to determine if sld3 gets modified in response to DNA damage
2. In this experiment: treated asynchronous yeast cells w either no drug, genotoxic drugs (cause DNA damage), or nocodazole (causes arrest at spindle checkpoint)… probed against sld3-3flag to assess mobility
3. Results: genotoxic drugs that cause DNA damage modify sld3 (increases its molecular weight)
   3a. Figure 1b: shows this modification is phosphorylation

Question 5

Figure 1C

Answer 5

1. Question: what kinase phosphorylated sld3 in response to DNA damage
2. What they do: researchers immunoblot many defective genes involved in DNA damage pathway and induce damage via MMS
3. Results: as shown, mec1 and rad53 are the only genes that change the mobility of sld3 (it doesn’t get phosphorylated when they are defected); therefore mec1 and rad53 are involved in the phosphorylation of sld3 when DNA damage occurs

Question 6

Figure 2A

Answer 6

1. Objective: see whether mutations in Sld3 and dbf4 preventing phosphorylation would eliminate damage-induced phosphorylation
2. Results: The SLD3-m25= no phosphorylation upon MMS 2a. Dbf4’s phosphorylation was not completely abolished in dbf4-m25, other kinases might also phosphorylate Dbf4?
3. Significance: confirmed that the phosphorylation of Sld3 is dependent on specific phosphorylation sites targeted by Rad53. The partial phosphorylation of Dbf4 suggests a more complex regulation involving additional kinases
4. This supports the hypothesis that both Sld3 and Dbf4 phosphorylation play key roles in the DNA damage response to prevent late origin firing

Question 7

Figure 2C

Answer 7

1. Objective: assess the functional consequence of SLD3-m25 and dbf4-m25 on the progression of the S phase during DNA damage.
2. Results: The SLD3-m25 dbf4-m25 double mutant progressed through the S phase faster than the wild-type and single mutants in the presence of MMS. =inappropriately fired late replication origins despite DNA damage
3. Significance: These results indicate that phosphorylation of both Sld3 and Dbf4 is crucial for the intra-S-phase checkpoint to function properly