My note on paper: A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin

Paper: A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin
(10.1016/j.dnarep.2006.05.045)
S. cerevisiae;
Rad18 function in PRR consist of
1.error-free damage bypass (Rad30 -Poln)
2.error-prone damage bypass (Rev3/7 -Pole)

DT40;
RAD18(-/-) cell
1. hypersensitivity to CPT but not RAD30(-/-)+REV3(-/-)
2. higher levels of H2Ax phosphorylation
3. higher in chromosomal aberration; gaps and breaks (after exposure to CPT)
4. no function in elongation without stimulant; but with CPT it showed more break

In conclusion;
this paper showed the role of RAD18 in replication fork when there is the lesion generate by CPT

Context:
CPT inhibit topoisomerase-I which is required for unsolve secondary structure during DNA replication

Inhibition of CPT can cause the db strand break during replication, therefore, initiate the ATR/ATM dependent phosphorylation of H2Ax.

There is evident showed DSB generated by CPT - being repair by HR.

In yeast study;
1. Show Rad6 (E2) bind Rad18 and play role in PRR
2. Rad6/Rad18 complex catalyzes monoubiquitination of PCNA -- involve in translesion synthesis
3. If monoubiquitination of PCNA turning to polyubiquitination of PCNA by  Mms2/Ubc13/Rad5 cpx -- initiate template switching DNA synthesis

The result from budding yeast is not correlated well in higher eukaryote on the PRR ex. mm2 in yeast hypersensitivity to DNA damage whereas in DT40 does not

no translesion DNA polymerase affect on Rad18-dependent CPT tolerance

Result:
*Hypersensitivity of DT40-Rad18(-/-) to CPT
1. show moderate sense to 4NQC (mimic to UV) but hypersensitive to CPT

*Role of TLS polymerase (related to Rad18) in CPT-tolerance
1. no evidence that could play role in CPT-tolerance

*Degradation of DNA after brief exposure to CPT (10 nM)
1. growth rate slower when under CPT treated
2. not affect on the elongation rate as indicated by alkaline sucrose density gradient
3. WT fixed the break better than Rad18(-/-) when induced by CPT

Bc RAD18 is knockout;
1. exclude the possibility of RAD18(-/-) affect other mutations causing sensitivity to CPT -- they did with 4 independently clones
2. transfer back ggRad18 regains the activity

Degradation of DNA after induced by brief CPT exposure;
1. Rad18(-/-) show slower growth rate when brief exposure to CPT

*Accumulation of DSB in Rad18(-/-) in the presence of CPT
1.Report showed H2Ax-P appears in human cell when induced by CPT
2.Measure the level of H2A-P after exposure of various of CPT; H2Ax forms much higher in Rad18(-/-)
3.More chromosomal gap and break found more in the Rad18(-/-) under CPT-treated, and also more than chromatid aberrations

CPT can induce strand break in both parental and newly synthesized strand (not by apoptosis induction as indicated by flow as well as the 100-bp ladder pattern)

- Hypersensitivity of Rad18(-/-) toward CPT is not explained by PRR (translesion synthesis or template switch DNA synthesis)
- Instead, result from abnormal replication which can be seen the newly synthesized DNA as well as the degradation of parental strand
- Proposed that newly replicated duplex is degraded by nuclease as shown by H2Ax (no. of DSB) and chromosome aberration (colcemid)