Note: Genetic dissection of vertebrate 53BP1: A major role in non-homologous end joining of DNA double strand breaks
Note: Genetic dissection of vertebrate 53BP1: A major role in non-homologous end joining of DNA double strand breaks
mutation of 53BP1 in mammalian causing
1.cellular sensitivity toward radiation.
2.defect in checkpoint
But DT40-53BP1(-/-)
1.intra-S phase checkpoint was normal
2.G2-M checkpoint was normal
once there is the DSB -- 53BP1 is the earliest protein to recruit and form foci.
In mammalian, defect in 53BP1 causes the cell cycle delays after the damage.
The sequence of NHEJ;
1. Ku70/80
2.DNA-PK --> phosphorylate Artemis nuclease
Artemis in IR break fixing is required more studies.
Late S-G2;
HR and NHEJ play role in "IR-induced" double strand break
G1-early S;
NHEJ is dominant
AA from 203-1716 was removed.
Sensitivity toward irradiation with asynchronously growing 53BP1-/- -->biphasic pattern.
53BP1(-/-) also increased the rate of HR --> this mean that it involves in the beginning step which HR competes with NHEJ (in case of DT40).
CPT inhibit topo-I which generate SSB but when entering S-phase --> generate the DSB during the replication (fix by HR). VP16 inhibits topo-II -->generate DSB (fix by NHEJ).
Analyze G2-M damage checkpoint -->mitotic index was measured by "phospho-histone 3 staining".
To see the checkpoint at intra-S phase --> using the thymidine incorporation assay.
They conclude that 53BP1 involves in NHEJ since the phenotype is pretty much the same with Ku70. There is biphasic phenotype when exposing to IR; 1.sensitive toward IR and 2.increasing dose become resistant -- if performing cellular synchronize -- G1 will be the most sensitive and S-G2 won't.
Epistatic relationship means 53BP1 has the affect on Ku70 -- if 53BP1 defect no phenotype of Ku70 is shown but if there is 53BP1 but lack of Ku70, Ku70-/- will show phenotype as 53BP1-/-.
They authors mentioned high-dose irradiation reflect the sensitivity in G2 phase cell (in asynchronous population). Ku suppresses HR after there is DSB (actually, I think it also relied on the physical DSB generated at the lesion -- and I think each genotoxic challenge would generate different physical break -- how Ku recognize the DNA end structure?)
The authors pointed out why 53BP1 might have less affect in suppress HR -- at the higher dose of IR - it showed the resistant almost to the WT but not beyond comparing to Ku70-/-.
No significant in vivo affect on the re-ligation of linearized plasmid.
Therefore, 53BP1 proposed to play role in the sub-pathway of NHEJ to fix DSB.
If there is the redundancy -- meaning that the phenotype of one particular gene will be hard to observe.
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