Abstract:
:Laser microirradiation is a powerful tool for real-time single-cell analysis of the DNA damage response (DDR). It is often found, however, that factor recruitment or modification profiles vary depending on the laser system employed. This is likely due to an incomplete understanding of how laser conditions/dosages affect the amounts and types of damage and the DDR. We compared different irradiation conditions using a femtosecond near-infrared laser and found distinct damage site recruitment thresholds for 53BP1 and TRF2 correlating with the dose-dependent increase of strand breaks and damage complexity. Low input-power microirradiation that induces relatively simple strand breaks led to robust recruitment of 53BP1 but not TRF2. In contrast, increased strand breaks with complex damage including crosslinking and base damage generated by high input-power microirradiation resulted in TRF2 recruitment to damage sites with no 53BP1 clustering. We found that poly(ADP-ribose) polymerase (PARP) activation distinguishes between the two damage states and that PARP activation is essential for rapid TRF2 recruitment while suppressing 53BP1 accumulation at damage sites. Thus, our results reveal that careful titration of laser irradiation conditions allows induction of varying amounts and complexities of DNA damage that are gauged by differential PARP activation regulating protein assembly at the damage site.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Saquilabon Cruz GM,Kong X,Silva BA,Khatibzadeh N,Thai R,Berns MW,Yokomori Kdoi
10.1093/nar/gkv976subject
Has Abstractpub_date
2016-02-18 00:00:00pages
e27issue
3eissn
0305-1048issn
1362-4962pii
gkv976journal_volume
44pub_type
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