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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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Mus81-Mms4 prefers duplex DNA flush to a joint molecule branch point. (A) Sample gels. Mus81-Mms4 activity was tested on DNA joint molecules related to a 3′-flapped structure, varied only by the 5′ end position of upstream duplex DNA. Duplex arm position was retreated from the branch point by 9 nt, 4 nt, 3 nt, 2 nt and 1 nt, and advanced from the branch point to a 5′ flap by 1 nt, 2 nt, 3 nt and 6 nt. Substrate concentration was defined by nonradiolabeled joint molecule fixed at 50 nM, and heterodimer was titrated at 0, 5, 10, 20, 50 and 100 nM heterodimer. Reactions were incubated at 30°C for 30 min and resolved by electrophoresis on native 10% PAGE–TBE gels, 100 V, 65 min. (B) Quantitation of results in (A), expressed as percent substrate cleaved during the nuclease assay. Incision at alternative sites occurs at high heterodimer concentrations (50 and 100 nM heterodimer) on structures with a 9-nt gap or 6-nt 5′-flap, indicated on the graph by pale bars. (C) Results in (A) and (B) under assay conditions where Mus81-Mms4 is limiting relative to substrate (5 nM heterodimer). The means and standard error of three independent assays are plotted.
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Figure 1: Mus81-Mms4 prefers duplex DNA flush to a joint molecule branch point. (A) Sample gels. Mus81-Mms4 activity was tested on DNA joint molecules related to a 3′-flapped structure, varied only by the 5′ end position of upstream duplex DNA. Duplex arm position was retreated from the branch point by 9 nt, 4 nt, 3 nt, 2 nt and 1 nt, and advanced from the branch point to a 5′ flap by 1 nt, 2 nt, 3 nt and 6 nt. Substrate concentration was defined by nonradiolabeled joint molecule fixed at 50 nM, and heterodimer was titrated at 0, 5, 10, 20, 50 and 100 nM heterodimer. Reactions were incubated at 30°C for 30 min and resolved by electrophoresis on native 10% PAGE–TBE gels, 100 V, 65 min. (B) Quantitation of results in (A), expressed as percent substrate cleaved during the nuclease assay. Incision at alternative sites occurs at high heterodimer concentrations (50 and 100 nM heterodimer) on structures with a 9-nt gap or 6-nt 5′-flap, indicated on the graph by pale bars. (C) Results in (A) and (B) under assay conditions where Mus81-Mms4 is limiting relative to substrate (5 nM heterodimer). The means and standard error of three independent assays are plotted.

Mentions: Unlike FEN1, Mus81-Mms4 activity is reduced on a dually flapped substrate (Figure 1). Of a panel of substrates related to the 3′-flapped joint molecule, the structure with duplex DNA flush to the branch point is processed most effectively. Mus81-Mms4 demands that the backbone discontinuity at the substrate branch point is unaltered by nucleotide additions to the deoxyribose at the 5′ position of the discontinuity that would overlap the 3′-flap. Mus81-Mms4 is more sensitive to extensions at the 5′ position of the nick than to retreat of the 5′ position from the branch point. Advancing duplex DNA from the branch point by 1–2 nt (which generates a short 5′ overhang) impairs substrate processing approximately ∼1.5–2.5-fold more than retreating duplex DNA from the branch point by 1–2 nt (which generates a small ssDNA gap) (Figure 1C). The overall DNA binding on these 3′-flap variants appears not considerably changed, as KM is not significantly altered when the duplex DNA is retreated or advanced by 1 nt from the branch point, and is nearly unaltered when the DNA is advanced or retreated by 2 nt (Figure 2). Instead, the processing impairment on these substrates is explained by a reduction in turnover. Altering the position of the duplex end by as little as one nucleotide reduces kcat, a reduction of 2.7-fold with a 1-nt flap, 5-fold with a 2-nt flap and nearly 25-fold with a 3-nt flap. Advancing the upstream duplex end as little as a single nucleotide of ssDNA reduces substrate processing by nearly 2-fold, and further advancing the upstream end into a 2-nt 5′ flap reduces substrate processing by up to 4-fold (Supplementary Table 1, Figure 2). We conclude that Mus81-Mms4 behaves very differently from FEN1 in its kinetic response to a dually flapped substrate. The -9-nt substrate (Figure 1A) showed evidence for an additional, novel cleavage site by Msu81-Mms4, which was mapped together with the cleavage sites of other substrates (see below; Figures 5 and 6).


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)

Mus81-Mms4 prefers duplex DNA flush to a joint molecule branch point. (A) Sample gels. Mus81-Mms4 activity was tested on DNA joint molecules related to a 3′-flapped structure, varied only by the 5′ end position of upstream duplex DNA. Duplex arm position was retreated from the branch point by 9 nt, 4 nt, 3 nt, 2 nt and 1 nt, and advanced from the branch point to a 5′ flap by 1 nt, 2 nt, 3 nt and 6 nt. Substrate concentration was defined by nonradiolabeled joint molecule fixed at 50 nM, and heterodimer was titrated at 0, 5, 10, 20, 50 and 100 nM heterodimer. Reactions were incubated at 30°C for 30 min and resolved by electrophoresis on native 10% PAGE–TBE gels, 100 V, 65 min. (B) Quantitation of results in (A), expressed as percent substrate cleaved during the nuclease assay. Incision at alternative sites occurs at high heterodimer concentrations (50 and 100 nM heterodimer) on structures with a 9-nt gap or 6-nt 5′-flap, indicated on the graph by pale bars. (C) Results in (A) and (B) under assay conditions where Mus81-Mms4 is limiting relative to substrate (5 nM heterodimer). The means and standard error of three independent assays are plotted.
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Related In: Results  -  Collection

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Figure 1: Mus81-Mms4 prefers duplex DNA flush to a joint molecule branch point. (A) Sample gels. Mus81-Mms4 activity was tested on DNA joint molecules related to a 3′-flapped structure, varied only by the 5′ end position of upstream duplex DNA. Duplex arm position was retreated from the branch point by 9 nt, 4 nt, 3 nt, 2 nt and 1 nt, and advanced from the branch point to a 5′ flap by 1 nt, 2 nt, 3 nt and 6 nt. Substrate concentration was defined by nonradiolabeled joint molecule fixed at 50 nM, and heterodimer was titrated at 0, 5, 10, 20, 50 and 100 nM heterodimer. Reactions were incubated at 30°C for 30 min and resolved by electrophoresis on native 10% PAGE–TBE gels, 100 V, 65 min. (B) Quantitation of results in (A), expressed as percent substrate cleaved during the nuclease assay. Incision at alternative sites occurs at high heterodimer concentrations (50 and 100 nM heterodimer) on structures with a 9-nt gap or 6-nt 5′-flap, indicated on the graph by pale bars. (C) Results in (A) and (B) under assay conditions where Mus81-Mms4 is limiting relative to substrate (5 nM heterodimer). The means and standard error of three independent assays are plotted.
Mentions: Unlike FEN1, Mus81-Mms4 activity is reduced on a dually flapped substrate (Figure 1). Of a panel of substrates related to the 3′-flapped joint molecule, the structure with duplex DNA flush to the branch point is processed most effectively. Mus81-Mms4 demands that the backbone discontinuity at the substrate branch point is unaltered by nucleotide additions to the deoxyribose at the 5′ position of the discontinuity that would overlap the 3′-flap. Mus81-Mms4 is more sensitive to extensions at the 5′ position of the nick than to retreat of the 5′ position from the branch point. Advancing duplex DNA from the branch point by 1–2 nt (which generates a short 5′ overhang) impairs substrate processing approximately ∼1.5–2.5-fold more than retreating duplex DNA from the branch point by 1–2 nt (which generates a small ssDNA gap) (Figure 1C). The overall DNA binding on these 3′-flap variants appears not considerably changed, as KM is not significantly altered when the duplex DNA is retreated or advanced by 1 nt from the branch point, and is nearly unaltered when the DNA is advanced or retreated by 2 nt (Figure 2). Instead, the processing impairment on these substrates is explained by a reduction in turnover. Altering the position of the duplex end by as little as one nucleotide reduces kcat, a reduction of 2.7-fold with a 1-nt flap, 5-fold with a 2-nt flap and nearly 25-fold with a 3-nt flap. Advancing the upstream duplex end as little as a single nucleotide of ssDNA reduces substrate processing by nearly 2-fold, and further advancing the upstream end into a 2-nt 5′ flap reduces substrate processing by up to 4-fold (Supplementary Table 1, Figure 2). We conclude that Mus81-Mms4 behaves very differently from FEN1 in its kinetic response to a dually flapped substrate. The -9-nt substrate (Figure 1A) showed evidence for an additional, novel cleavage site by Msu81-Mms4, which was mapped together with the cleavage sites of other substrates (see below; Figures 5 and 6).

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
Related in: MedlinePlus