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Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro

The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we...

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Autores principales: Purohit, Nupur K., Robu, Mihaela, Shah, Rashmi G., Geacintov, Nicholas E., Shah, Girish M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709520/
https://www.ncbi.nlm.nih.gov/pubmed/26753915
http://dx.doi.org/10.1038/srep19020
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author Purohit, Nupur K.
Robu, Mihaela
Shah, Rashmi G.
Geacintov, Nicholas E.
Shah, Girish M.
author_facet Purohit, Nupur K.
Robu, Mihaela
Shah, Rashmi G.
Geacintov, Nicholas E.
Shah, Girish M.
author_sort Purohit, Nupur K.
collection PubMed
description The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from −12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA.
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spelling pubmed-47095202016-01-20 Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro Purohit, Nupur K. Robu, Mihaela Shah, Rashmi G. Geacintov, Nicholas E. Shah, Girish M. Sci Rep Article The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from −12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA. Nature Publishing Group 2016-01-12 /pmc/articles/PMC4709520/ /pubmed/26753915 http://dx.doi.org/10.1038/srep19020 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Purohit, Nupur K.
Robu, Mihaela
Shah, Rashmi G.
Geacintov, Nicholas E.
Shah, Girish M.
Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro
title Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro
title_full Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro
title_fullStr Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro
title_full_unstemmed Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro
title_short Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro
title_sort characterization of the interactions of parp-1 with uv-damaged dna in vivo and in vitro
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709520/
https://www.ncbi.nlm.nih.gov/pubmed/26753915
http://dx.doi.org/10.1038/srep19020
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