title: Femtosecond near-infrared laser microirradiation reveals a crucial role for PARP signaling on factor assemblies at DNA damage sites.
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.
authors.affiliation: Beckman Laser Institute and Medical Clinic, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA.
authors.forename: Gladys Mae
authors.initials: GM
authors.lastname: Saquilabon Cruz
authors.affiliation: Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA 92697-1700, USA.
authors.forename: Xiangduo
authors.initials: X
authors.lastname: Kong
authors.affiliation: Beckman Laser Institute and Medical Clinic, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, CA 92617, USA.
authors.forename: Bárbara Alcaraz
authors.initials: BA
authors.lastname: Silva
authors.affiliation: Beckman Laser Institute and Medical Clinic, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA.
authors.forename: Nima
authors.initials: N
authors.lastname: Khatibzadeh
authors.affiliation: Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA 92697-1700, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Thai
authors.affiliation: Beckman Laser Institute and Medical Clinic, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, CA 92617, USA Department of Biomedical Engineering and Surgery, University of California, Irvine, CA 92617, USA mwberns@uci.edu.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Berns
authors.affiliation: Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA 92697-1700, USA kyokomor@uci.edu.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Yokomori
mesh_terms: D002460:Cell Line; D004249:DNA Damage; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D007834:Lasers; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D015398:Signal Transduction; D035341:Telomeric Repeat Binding Protein 2 / Q000235:genetics; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C504753:TERF2 protein, human; C498301:TP53BP1 protein, human; D035341:Telomeric Repeat Binding Protein 2; D000071857:Tumor Suppressor p53-Binding Protein 1; D011065:Poly(ADP-ribose) Polymerases