title: A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells.
abstract: DNA-protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA-protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Jordan
authors: J
authors: Woodrick
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Suhani
authors: S
authors: Gupta
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Pooja
authors: P
authors: Khatkar
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Kalpana
authors: K
authors: Dave
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Darya
authors: D
authors: Levashova
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Sujata
authors: S
authors: Choudhury
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Hadi
authors: H
authors: Elias
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Tapas
authors: T
authors: Saha
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States.
authors: Susette
authors: S
authors: Mueller
authors: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, United States. Electronic address: rr228@georgetown.edu.
authors: Rabindra
authors: R
authors: Roy
mesh_terms: D002460:Cell Line; D018736:DNA Adducts / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D045585:Flap Endonucleases / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D014162:Transfection
chemical_list: D018736:DNA Adducts; C000607892:LIG1 protein, human; D019951:DNA Polymerase beta; D045585:Flap Endonucleases; C468317:FEN1 protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases; D000072481:DNA Ligase ATP
keywords: Abasic sites; Base excision repair; Co-localization; DNA–protein interaction; Repair intermediate