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A junction branch point adjacent to a DNA backbone nick directs substrate cleavage by Saccharomyces cerevisiae Mus81-Mms4.

Ehmsen KT, Heyer WD - Nucleic Acids Res. (2009)

Bottom Line: Here we test the activity of Mus81-Mms4 on dually flapped substrates and find that in contrast to FEN1/Rad27, Mus81-Mms4 activity is impaired on such substrates, resulting in cleavage products that do not allow direct religation.We conclude that Mus81-Mms4, unlike FEN1/Rad27, does not prefer dually flapped substrates and is unlikely to function as a 3'-flapase counterpart to the 5'-flapase activity of FEN1/Rad27.These findings underscore the significance of a nick adjacent to a branch point for Mus81-Mms4 incision.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of California, Davis, CA 95616-8665, USA.

ABSTRACT
The DNA structure-selective endonuclease Mus81-Mms4/Eme1 incises a number of nicked joint molecule substrates in vitro. 3'-flaps are an excellent in vitro substrate for Mus81-Mms4/Eme1. Mutants in MUS81 are synthetically lethal with mutations in the 5'-flap endonuclease FEN1/Rad27 in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Considering the possibility for isoenergetic interconversion between 3'- and 5'- flaps, these data are consistent with the hypothesis that Mus81-Mms4/Eme1 acts on 3'-flaps in vivo. FEN1/Rad27 prefers dually flapped substrates and cleaves in a way that allows direct ligation of the resulting nick in the product duplex. Here we test the activity of Mus81-Mms4 on dually flapped substrates and find that in contrast to FEN1/Rad27, Mus81-Mms4 activity is impaired on such substrates, resulting in cleavage products that do not allow direct religation. We conclude that Mus81-Mms4, unlike FEN1/Rad27, does not prefer dually flapped substrates and is unlikely to function as a 3'-flapase counterpart to the 5'-flapase activity of FEN1/Rad27. We further find that joint molecule incision by Mus81-Mms4 occurs in a fashion determined by the branch point, regardless of the position of an upstream duplex end. These findings underscore the significance of a nick adjacent to a branch point for Mus81-Mms4 incision.

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Mapping of Mus81-Mms4 incision sites at high protein to substrate ratio. Denaturing urea–PAGE analysis of substrate incision sites on RF-like, nXO12, D-loop (DL) and five 3′-FL-related structures. Assays were performed with 50 nM His10-FLAG-Mus81/GST-Mms4, 50 nM substrate, 30 min at 30°C. Where indicated, reactions were then supplemented with 0.5 mM ATP/3 mM Mg(OAc)2 and 10 U T4 DNA ligase, with incubation at room temperature for 15 min. All assays were terminated by boiling at 95°C, 2 min, with immediate transfer to ice. ‘L’ represents an oligonucleotide size ladder. The scheme on the right side of the gel illustrates the substrate and cleavage site; the star denotes the position of the 5′ label.
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Figure 5: Mapping of Mus81-Mms4 incision sites at high protein to substrate ratio. Denaturing urea–PAGE analysis of substrate incision sites on RF-like, nXO12, D-loop (DL) and five 3′-FL-related structures. Assays were performed with 50 nM His10-FLAG-Mus81/GST-Mms4, 50 nM substrate, 30 min at 30°C. Where indicated, reactions were then supplemented with 0.5 mM ATP/3 mM Mg(OAc)2 and 10 U T4 DNA ligase, with incubation at room temperature for 15 min. All assays were terminated by boiling at 95°C, 2 min, with immediate transfer to ice. ‘L’ represents an oligonucleotide size ladder. The scheme on the right side of the gel illustrates the substrate and cleavage site; the star denotes the position of the 5′ label.

Mentions: Mus81-Mms4 incises joint molecules at the branch point, adjacent to a phosphodiester backbone discontinuity. Quantitation of incision sites in Figure 5. Substrate branch point position is indicated by a vertical gray bar in all substrate schematics; the phosphodiester bond between the fourth and fifth nucleotides 5′ to the branch point is indicated by a vertical yellow bar.


A junction branch point adjacent to a DNA backbone nick directs substrate cleavage by Saccharomyces cerevisiae Mus81-Mms4.

Ehmsen KT, Heyer WD - Nucleic Acids Res. (2009)

Mapping of Mus81-Mms4 incision sites at high protein to substrate ratio. Denaturing urea–PAGE analysis of substrate incision sites on RF-like, nXO12, D-loop (DL) and five 3′-FL-related structures. Assays were performed with 50 nM His10-FLAG-Mus81/GST-Mms4, 50 nM substrate, 30 min at 30°C. Where indicated, reactions were then supplemented with 0.5 mM ATP/3 mM Mg(OAc)2 and 10 U T4 DNA ligase, with incubation at room temperature for 15 min. All assays were terminated by boiling at 95°C, 2 min, with immediate transfer to ice. ‘L’ represents an oligonucleotide size ladder. The scheme on the right side of the gel illustrates the substrate and cleavage site; the star denotes the position of the 5′ label.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665226&req=5

Figure 5: Mapping of Mus81-Mms4 incision sites at high protein to substrate ratio. Denaturing urea–PAGE analysis of substrate incision sites on RF-like, nXO12, D-loop (DL) and five 3′-FL-related structures. Assays were performed with 50 nM His10-FLAG-Mus81/GST-Mms4, 50 nM substrate, 30 min at 30°C. Where indicated, reactions were then supplemented with 0.5 mM ATP/3 mM Mg(OAc)2 and 10 U T4 DNA ligase, with incubation at room temperature for 15 min. All assays were terminated by boiling at 95°C, 2 min, with immediate transfer to ice. ‘L’ represents an oligonucleotide size ladder. The scheme on the right side of the gel illustrates the substrate and cleavage site; the star denotes the position of the 5′ label.
Mentions: Mus81-Mms4 incises joint molecules at the branch point, adjacent to a phosphodiester backbone discontinuity. Quantitation of incision sites in Figure 5. Substrate branch point position is indicated by a vertical gray bar in all substrate schematics; the phosphodiester bond between the fourth and fifth nucleotides 5′ to the branch point is indicated by a vertical yellow bar.

Bottom Line: Here we test the activity of Mus81-Mms4 on dually flapped substrates and find that in contrast to FEN1/Rad27, Mus81-Mms4 activity is impaired on such substrates, resulting in cleavage products that do not allow direct religation.We conclude that Mus81-Mms4, unlike FEN1/Rad27, does not prefer dually flapped substrates and is unlikely to function as a 3'-flapase counterpart to the 5'-flapase activity of FEN1/Rad27.These findings underscore the significance of a nick adjacent to a branch point for Mus81-Mms4 incision.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of California, Davis, CA 95616-8665, USA.

ABSTRACT
The DNA structure-selective endonuclease Mus81-Mms4/Eme1 incises a number of nicked joint molecule substrates in vitro. 3'-flaps are an excellent in vitro substrate for Mus81-Mms4/Eme1. Mutants in MUS81 are synthetically lethal with mutations in the 5'-flap endonuclease FEN1/Rad27 in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Considering the possibility for isoenergetic interconversion between 3'- and 5'- flaps, these data are consistent with the hypothesis that Mus81-Mms4/Eme1 acts on 3'-flaps in vivo. FEN1/Rad27 prefers dually flapped substrates and cleaves in a way that allows direct ligation of the resulting nick in the product duplex. Here we test the activity of Mus81-Mms4 on dually flapped substrates and find that in contrast to FEN1/Rad27, Mus81-Mms4 activity is impaired on such substrates, resulting in cleavage products that do not allow direct religation. We conclude that Mus81-Mms4, unlike FEN1/Rad27, does not prefer dually flapped substrates and is unlikely to function as a 3'-flapase counterpart to the 5'-flapase activity of FEN1/Rad27. We further find that joint molecule incision by Mus81-Mms4 occurs in a fashion determined by the branch point, regardless of the position of an upstream duplex end. These findings underscore the significance of a nick adjacent to a branch point for Mus81-Mms4 incision.

Show MeSH