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Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage.
Koczor, Christopher A; Saville, Kate M; Al-Rahahleh, Rasha Q; Andrews, Joel F; Li, Jianfeng; Sobol, Robert W.
Afiliação
  • Koczor CA; Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
  • Saville KM; Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL, USA.
  • Al-Rahahleh RQ; Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
  • Andrews JF; Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL, USA.
  • Li J; Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
  • Sobol RW; Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL, USA.
Methods Mol Biol ; 2609: 43-59, 2023.
Article em En | MEDLINE | ID: mdl-36515828
Poly(ADP-ribose) (PAR), catalyzed by members of the poly(ADP-ribose) polymerase family of enzymes, is a posttranslational modification with a critical role in most mechanisms of DNA repair. Upon activation of poly(ADP-ribose) polymerase isoforms 1 and 2 (PARP-1 and PARP-2), the proteins of the base excision repair (BER) and single-strand break repair (SSBR) pathways form DNA lesion-dependent, transient complexes to facilitate repair. PAR is central to the temporal dynamics of BER/SSBR complex assembly and disassembly. To enhance cellular PAR analysis, we developed LivePAR, a fluorescently tagged PAR-binding fusion protein and genetically encoded imaging probe for live cell, quantitative analysis of PAR in mammalian cells. LivePAR has the advantage that it enables real-time imaging of PAR formation in cells and significantly overcomes limitations of immunocytochemistry for PAR analysis. This chapter describes the protocols needed to develop cells expressing LivePAR or EGFP-tagged BER proteins and to evaluate laser-induced formation of PAR and comparison to the assembly of the BER proteins XRCC1 and DNA polymerase-ß.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Poli Adenosina Difosfato Ribose / Poli(ADP-Ribose) Polimerases Limite: Animals Idioma: En Ano de publicação: 2023 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Poli Adenosina Difosfato Ribose / Poli(ADP-Ribose) Polimerases Limite: Animals Idioma: En Ano de publicação: 2023 Tipo de documento: Article