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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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Comparison of Mus81-Mms4 kinetic values in relation to DNA advanced or retreated from the branch point. Kinetic parameters on DNA joint molecules related to a 3′-flapped substrate are plotted in direct comparison to the 3′-flapped structure. Kinetic analysis was performed on structures with the upstream 5′ duplex position retreated from the branch point by 1 nt (–1 nt) and 2 nt (–2 nt), or with the upstream duplex absent (Y); and on structures with the upstream 5′ duplex position advanced from the branch point to form a short 5′ flap of 1 nt (+1 nt), 2 nt (+2 nt) or 3 nt (+3 nt). (A)kcat (nM min−1), (B)KM (nM), (C) selectivity coefficient kcat/KM (nM−1 min−1). The data for the Y substrate are from Ehmsen and Heyer (13).
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Figure 2: Comparison of Mus81-Mms4 kinetic values in relation to DNA advanced or retreated from the branch point. Kinetic parameters on DNA joint molecules related to a 3′-flapped substrate are plotted in direct comparison to the 3′-flapped structure. Kinetic analysis was performed on structures with the upstream 5′ duplex position retreated from the branch point by 1 nt (–1 nt) and 2 nt (–2 nt), or with the upstream duplex absent (Y); and on structures with the upstream 5′ duplex position advanced from the branch point to form a short 5′ flap of 1 nt (+1 nt), 2 nt (+2 nt) or 3 nt (+3 nt). (A)kcat (nM min−1), (B)KM (nM), (C) selectivity coefficient kcat/KM (nM−1 min−1). The data for the Y substrate are from Ehmsen and Heyer (13).

Mentions: Assays were performed as described by Ehmsen and Heyer (13). Heterodimer was diluted in standard enzyme diluent (SED: 10 mM Tris–HCl, pH 7.5, 0.5 mg/ml BSA) to an appropriate 10× reaction stock. Reactions were performed in buffer containing 25 mM HEPES pH 7.5, 3 mM MgOAc2, 1 mM DTT and 0.1 μg/ml BSA. For protein titrations on 3′-flap variants, three independent trials were performed with substrate concentration fixed at 50 nM and Mus81-Mms4 added to 5, 10, 20, 50 and 100 nM heterodimer. Reactions without protein were supplemented with standard enzyme diluent (SED: 10 mM Tris–HCl, pH7.5, 0.5 mg with pH7.5, 0.5 mg BSA). Reactions were pre-incubated in a 30°C water bath for 10 min, and were initiated by addition of an appropriate heterodimer dilution in SED with gentle mixing, followed by a 30-min incubation. Reactions were quenched by addition of 2 μl nuclease stop mix (200 mM EDTA, 2.5% SDS, 10 mg/ml proteinase K). Reactions were normalized for radioactivity and processed for electrophoresis as described for kinetic assays below. For kinetic analysis, at least three independent trials were performed for the substrates indicated in Figure 2 at 0.5, 1, 2.5, 5, 10, 20, 50 and 100 nM substrate and enzyme fixed at 5 nM, with all substrate concentrations processed in parallel. Reactions were preincubated in a 30°C water bath for 10 min, and were initiated by addition of 1 µl 50 nM heterodimer with gentle mixing. 0.5 µl aliquots were removed from each [substrate] reaction at 3, 6, 10, 15, 20, 30, 45 and 60 min and quenched immediately into pre-aliquoted 0.5 µl volumes of 0.5× nuclease stop mix. At each time point, 9 µl DNA loading dye (5% glycerol/bromophenol blue) were added and samples were electrophoresed by 10 × 20 cm native 10% TBE–PAGE at 100 V for 65 min. Gels were equilibrated in 5% glycerol or 3% glycerol/20% methanol for 30 min and vacuum-dried to Whatman® paper at 65°C, then exposed overnight to a phosphorimager screen. Reaction progress was quantified by Storm Phosphorimagery and ImageQuant software. Initial velocities were extrapolated from nonlinear regression curves defined by Graphpad Prism at 30 s reaction time. Michaelis–Menten analysis was performed using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com). Rates are expressed as nanomoles joint molecule substrate incised/minute, and where calculated for kcat, rates are expressed as number of joint molecule substrates incised per heterodimer molecule per minute.


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)

Comparison of Mus81-Mms4 kinetic values in relation to DNA advanced or retreated from the branch point. Kinetic parameters on DNA joint molecules related to a 3′-flapped substrate are plotted in direct comparison to the 3′-flapped structure. Kinetic analysis was performed on structures with the upstream 5′ duplex position retreated from the branch point by 1 nt (–1 nt) and 2 nt (–2 nt), or with the upstream duplex absent (Y); and on structures with the upstream 5′ duplex position advanced from the branch point to form a short 5′ flap of 1 nt (+1 nt), 2 nt (+2 nt) or 3 nt (+3 nt). (A)kcat (nM min−1), (B)KM (nM), (C) selectivity coefficient kcat/KM (nM−1 min−1). The data for the Y substrate are from Ehmsen and Heyer (13).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Comparison of Mus81-Mms4 kinetic values in relation to DNA advanced or retreated from the branch point. Kinetic parameters on DNA joint molecules related to a 3′-flapped substrate are plotted in direct comparison to the 3′-flapped structure. Kinetic analysis was performed on structures with the upstream 5′ duplex position retreated from the branch point by 1 nt (–1 nt) and 2 nt (–2 nt), or with the upstream duplex absent (Y); and on structures with the upstream 5′ duplex position advanced from the branch point to form a short 5′ flap of 1 nt (+1 nt), 2 nt (+2 nt) or 3 nt (+3 nt). (A)kcat (nM min−1), (B)KM (nM), (C) selectivity coefficient kcat/KM (nM−1 min−1). The data for the Y substrate are from Ehmsen and Heyer (13).
Mentions: Assays were performed as described by Ehmsen and Heyer (13). Heterodimer was diluted in standard enzyme diluent (SED: 10 mM Tris–HCl, pH 7.5, 0.5 mg/ml BSA) to an appropriate 10× reaction stock. Reactions were performed in buffer containing 25 mM HEPES pH 7.5, 3 mM MgOAc2, 1 mM DTT and 0.1 μg/ml BSA. For protein titrations on 3′-flap variants, three independent trials were performed with substrate concentration fixed at 50 nM and Mus81-Mms4 added to 5, 10, 20, 50 and 100 nM heterodimer. Reactions without protein were supplemented with standard enzyme diluent (SED: 10 mM Tris–HCl, pH7.5, 0.5 mg with pH7.5, 0.5 mg BSA). Reactions were pre-incubated in a 30°C water bath for 10 min, and were initiated by addition of an appropriate heterodimer dilution in SED with gentle mixing, followed by a 30-min incubation. Reactions were quenched by addition of 2 μl nuclease stop mix (200 mM EDTA, 2.5% SDS, 10 mg/ml proteinase K). Reactions were normalized for radioactivity and processed for electrophoresis as described for kinetic assays below. For kinetic analysis, at least three independent trials were performed for the substrates indicated in Figure 2 at 0.5, 1, 2.5, 5, 10, 20, 50 and 100 nM substrate and enzyme fixed at 5 nM, with all substrate concentrations processed in parallel. Reactions were preincubated in a 30°C water bath for 10 min, and were initiated by addition of 1 µl 50 nM heterodimer with gentle mixing. 0.5 µl aliquots were removed from each [substrate] reaction at 3, 6, 10, 15, 20, 30, 45 and 60 min and quenched immediately into pre-aliquoted 0.5 µl volumes of 0.5× nuclease stop mix. At each time point, 9 µl DNA loading dye (5% glycerol/bromophenol blue) were added and samples were electrophoresed by 10 × 20 cm native 10% TBE–PAGE at 100 V for 65 min. Gels were equilibrated in 5% glycerol or 3% glycerol/20% methanol for 30 min and vacuum-dried to Whatman® paper at 65°C, then exposed overnight to a phosphorimager screen. Reaction progress was quantified by Storm Phosphorimagery and ImageQuant software. Initial velocities were extrapolated from nonlinear regression curves defined by Graphpad Prism at 30 s reaction time. Michaelis–Menten analysis was performed using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com). Rates are expressed as nanomoles joint molecule substrate incised/minute, and where calculated for kcat, rates are expressed as number of joint molecule substrates incised per heterodimer molecule per minute.

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