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.
|School or Research Centre:||Birkbeck Schools and Research Centres > School of Science > Biological Sciences|
|Date Deposited:||24 Apr 2012 11:36|
|Last Modified:||17 Apr 2013 12:33|
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