Shining light on the response to repair intermediates in DNA of living cells
| Autor: | Ming-Lang Zhao, C. Jeff Tucker, Agnes K. Janoshazi, Rajendra Prasad, Erica L. Scappini, Julie K. Horton, Samuel H. Wilson |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
DNA Repair
DNA polymerase DNA damage Poly (ADP-Ribose) Polymerase-1 Biochemistry Article Dissociation (chemistry) Mice 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine PARP1 In vivo DNA-(Apurinic or Apyrimidinic Site) Lyase Animals Humans Molecular Biology Cells Cultured DNA Polymerase beta 030304 developmental biology 0303 health sciences biology Cell Biology Base excision repair Nuclear DNA Kinetics chemistry 030220 oncology & carcinogenesis biology.protein Biophysics DNA DNA Damage |
| Zdroj: | DNA Repair (Amst) |
| ISSN: | 1568-7864 |
| Popis: | Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase β (pol β). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol β in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol β. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect