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

Bowles M, Lally J, Fadden AJ, Mouilleron S, Hammonds T, McDonald NQ - Nucleic Acids Res. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.

ABSTRACT
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.

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Local sequence preferences at the XPF–ERCC1 incision site on the substrate affects the rate of cleavage. (A) Stem–loop structure showing the positions of substituted bases X and Y in the duplex. Xc and Yc are the complementary bases to X and Y, A is used complementary to U. (B) Kinetic data where X and Y were substituted with each of the four bases. Data are mean of triplicate samples, ±1 standard error. (C) Incisions produced by 4.27 nM XPF–ERCC1 on 5′ 6-FAM-labelled stem–loop substrates with bases at X and Y as indicated. Arrow shows cleavage product. Reactions were incubated for 15 min at 25°C and therefore did not run to completion. (D) Kinetic data where X and Y were substituted with the indicated bases. Data are mean of triplicate samples +/− standard error.
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gks284-F3: Local sequence preferences at the XPF–ERCC1 incision site on the substrate affects the rate of cleavage. (A) Stem–loop structure showing the positions of substituted bases X and Y in the duplex. Xc and Yc are the complementary bases to X and Y, A is used complementary to U. (B) Kinetic data where X and Y were substituted with each of the four bases. Data are mean of triplicate samples, ±1 standard error. (C) Incisions produced by 4.27 nM XPF–ERCC1 on 5′ 6-FAM-labelled stem–loop substrates with bases at X and Y as indicated. Arrow shows cleavage product. Reactions were incubated for 15 min at 25°C and therefore did not run to completion. (D) Kinetic data where X and Y were substituted with the indicated bases. Data are mean of triplicate samples +/− standard error.

Mentions: Stem–loops and oligonucleotides F1 and F3 (sequences described above and used to make the structures in Figure 3E as described below) were labelled on the 5′-terminus with [γ32P]ATP using T4 polynucleotide kinase (NEB) for 30 min at 37°C, and the reaction stopped by heating to 95°C for 3 min. F1 was mixed with 2 M excess of F2 for double-strand (duplex), and F4 for 5′-overhang. F3 was mixed with F2 for 3′-overhang, and F4 for splayed arm.


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

Bowles M, Lally J, Fadden AJ, Mouilleron S, Hammonds T, McDonald NQ - Nucleic Acids Res. (2012)

Local sequence preferences at the XPF–ERCC1 incision site on the substrate affects the rate of cleavage. (A) Stem–loop structure showing the positions of substituted bases X and Y in the duplex. Xc and Yc are the complementary bases to X and Y, A is used complementary to U. (B) Kinetic data where X and Y were substituted with each of the four bases. Data are mean of triplicate samples, ±1 standard error. (C) Incisions produced by 4.27 nM XPF–ERCC1 on 5′ 6-FAM-labelled stem–loop substrates with bases at X and Y as indicated. Arrow shows cleavage product. Reactions were incubated for 15 min at 25°C and therefore did not run to completion. (D) Kinetic data where X and Y were substituted with the indicated bases. Data are mean of triplicate samples +/− standard error.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401468&req=5

gks284-F3: Local sequence preferences at the XPF–ERCC1 incision site on the substrate affects the rate of cleavage. (A) Stem–loop structure showing the positions of substituted bases X and Y in the duplex. Xc and Yc are the complementary bases to X and Y, A is used complementary to U. (B) Kinetic data where X and Y were substituted with each of the four bases. Data are mean of triplicate samples, ±1 standard error. (C) Incisions produced by 4.27 nM XPF–ERCC1 on 5′ 6-FAM-labelled stem–loop substrates with bases at X and Y as indicated. Arrow shows cleavage product. Reactions were incubated for 15 min at 25°C and therefore did not run to completion. (D) Kinetic data where X and Y were substituted with the indicated bases. Data are mean of triplicate samples +/− standard error.
Mentions: Stem–loops and oligonucleotides F1 and F3 (sequences described above and used to make the structures in Figure 3E as described below) were labelled on the 5′-terminus with [γ32P]ATP using T4 polynucleotide kinase (NEB) for 30 min at 37°C, and the reaction stopped by heating to 95°C for 3 min. F1 was mixed with 2 M excess of F2 for double-strand (duplex), and F4 for 5′-overhang. F3 was mixed with F2 for 3′-overhang, and F4 for splayed arm.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.

ABSTRACT
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.

Show MeSH