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Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition

Bowles, M. and Lally, J. and Fadden, A.J. and Mouilleron, S. and Hammonds, T. and McDonald, Neil Q. (2012) Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition. Nucleic Acids Research 40 (13), e101. ISSN 0305-1048.

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The structure-specific endonuclease activity of the human XPF–ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF–ERCC1 cleaves DNA structures such as stem–loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem–loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF–ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3′-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix–hairpin–helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF–HLD shows a preference for binding stem–loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF–ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity.

Item Type: Article
School or Research Centre: Birkbeck Schools and Research Centres > School of Science > Biological Sciences
Depositing User: Administrator
Date Deposited: 24 Apr 2012 11:36
Last Modified: 17 Apr 2013 12:33

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