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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 incises joint molecules at the branch point, adjacent to a phosphodiester backbone discontinuity. Quantitation of incision sites in Figure 3. 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.
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Figure 4: Mus81-Mms4 incises joint molecules at the branch point, adjacent to a phosphodiester backbone discontinuity. Quantitation of incision sites in Figure 3. 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.

Mentions: To test the role of the upstream duplex in positioning Mus81-Mms4 incision, we determined the position of substrate incision on a number of DNA joint molecules. We incubated limiting heterodimer with excess substrate (10 nM heterodimer with 50 nM substrate; Figures 3 and 4) or equimolar heterodimer and substrate (50 nM heterodimer with 50 nM substrate; Figures 5 and 6) on nXO12 (nicked Holliday junction), RF-like (replication fork-like), DL (D-loop) and 3′-FL (3′-flap)-related structures, for 30 min at 30°C. Assays in the presence of limiting heterodimer were used to determine incision site positions under conditions that allow Mus81-Mms4 catalytic turnover; assays with stoichiometric quantities of heterodimer and substrate allowed the determination of incision site positions on an additional set of substrates that are too poorly incised for incision site quantitation under conditions of limiting heterodimer (specifically the 3′-flapped structures with 4-nt and 9-nt gaps, and 6-nt 5′-flap). We interpreted incision site positions by direct comparison of the incised oligonucleotide lengths to a radiolabeled ladder of oligonucleotide standards of known lengths (Figures 3 and 5). In all cases, incision takes place on the upper strand of the joint molecule (defined as the DNA backbone continuous with the 3′-flap in 3′-flapped structures), achieved by hydrolysis of the phosphodiester bond between the fourth and fifth nucleotides 5′ to the junction branch point (Figures 4 and 6). This is true whether the duplex DNA upstream of the branch point is advanced to a single-stranded 5′ DNA overhang or retreated to generate a small single-stranded DNA gap.


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 incises joint molecules at the branch point, adjacent to a phosphodiester backbone discontinuity. Quantitation of incision sites in Figure 3. 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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Mus81-Mms4 incises joint molecules at the branch point, adjacent to a phosphodiester backbone discontinuity. Quantitation of incision sites in Figure 3. 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.
Mentions: To test the role of the upstream duplex in positioning Mus81-Mms4 incision, we determined the position of substrate incision on a number of DNA joint molecules. We incubated limiting heterodimer with excess substrate (10 nM heterodimer with 50 nM substrate; Figures 3 and 4) or equimolar heterodimer and substrate (50 nM heterodimer with 50 nM substrate; Figures 5 and 6) on nXO12 (nicked Holliday junction), RF-like (replication fork-like), DL (D-loop) and 3′-FL (3′-flap)-related structures, for 30 min at 30°C. Assays in the presence of limiting heterodimer were used to determine incision site positions under conditions that allow Mus81-Mms4 catalytic turnover; assays with stoichiometric quantities of heterodimer and substrate allowed the determination of incision site positions on an additional set of substrates that are too poorly incised for incision site quantitation under conditions of limiting heterodimer (specifically the 3′-flapped structures with 4-nt and 9-nt gaps, and 6-nt 5′-flap). We interpreted incision site positions by direct comparison of the incised oligonucleotide lengths to a radiolabeled ladder of oligonucleotide standards of known lengths (Figures 3 and 5). In all cases, incision takes place on the upper strand of the joint molecule (defined as the DNA backbone continuous with the 3′-flap in 3′-flapped structures), achieved by hydrolysis of the phosphodiester bond between the fourth and fifth nucleotides 5′ to the junction branch point (Figures 4 and 6). This is true whether the duplex DNA upstream of the branch point is advanced to a single-stranded 5′ DNA overhang or retreated to generate a small single-stranded DNA gap.

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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