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Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures.

Kang YH, Munashingha PR, Lee CH, Nguyen TA, Seo YS - Nucleic Acids Res. (2011)

Bottom Line: Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase.For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap.We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Center for DNA Replication and Genome Instability, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.

ABSTRACT
Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.

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Mph1 DNA helicase activity on substrates containing two 5′-flaps. (A) Schematic models of Mph1 unwinding mechanism. (B) Helicase activity of Mph1 on substrates containing two 5′-flaps. Assays were performed for various incubation periods (1, 4, 10 and 25 min). f1, flap1; f2, flap2.
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gkr983-F3: Mph1 DNA helicase activity on substrates containing two 5′-flaps. (A) Schematic models of Mph1 unwinding mechanism. (B) Helicase activity of Mph1 on substrates containing two 5′-flaps. Assays were performed for various incubation periods (1, 4, 10 and 25 min). f1, flap1; f2, flap2.

Mentions: The sequences of oligonucleotides used for substrate preparations are listed in Table 1. Partial-duplex substrates were prepared as follows; one of the two oligonucleotides (10 pmol) in a partial-duplex DNA substrate was 5′-labeled with 3.3 pmol of [γ-32P] ATP and polynucleotide kinase according to the manufacturer's protocol. The labeled oligonucleotides were then annealed to the second oligonucleotide in 50 mM HEPES–NaOH/pH 7.5 and 200 mM NaCl, using a PCR machine (95°C: 5 min, 65°C: 30 min and −0.5°C/min to 25°C). For the preparation of flap-structured or nicked-duplex DNA substrates, one oligonucleotide was 5′-end labeled as described above and then annealed to the template and upstream oligonucleotides using a molar ratio of 1:2:4, respectively. To prepare DNA substrates containing two 5′-flaps (as shown in Figure 3), four-way junction (Figure 7) and replication fork-like substrates (fixed or partially movable double-strand three-way junction and regressed replication fork; shown in Figures 8A and 9), one of the oligonucleotides was labeled as described above and annealed with other three oligonucleotides at a molar ratio of 1:1:1:1. For the preparation of movable double-stranded three-way junction substrate (Figure 8B, C and D), two oligonucleotides 11 and 35 were first labeled and then annealed (1:1 ratio) separately to oligonucleotides 14 and 34, respectively. Subsequently, both partial-duplex DNAs were mixed together (1:1) and allowed to anneal for 12 h at 25°C. All annealed substrates were purified by 10% PAGE and the amount of recovered substrate was determined as previously described (14).Table 1.


Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures.

Kang YH, Munashingha PR, Lee CH, Nguyen TA, Seo YS - Nucleic Acids Res. (2011)

Mph1 DNA helicase activity on substrates containing two 5′-flaps. (A) Schematic models of Mph1 unwinding mechanism. (B) Helicase activity of Mph1 on substrates containing two 5′-flaps. Assays were performed for various incubation periods (1, 4, 10 and 25 min). f1, flap1; f2, flap2.
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Related In: Results  -  Collection

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gkr983-F3: Mph1 DNA helicase activity on substrates containing two 5′-flaps. (A) Schematic models of Mph1 unwinding mechanism. (B) Helicase activity of Mph1 on substrates containing two 5′-flaps. Assays were performed for various incubation periods (1, 4, 10 and 25 min). f1, flap1; f2, flap2.
Mentions: The sequences of oligonucleotides used for substrate preparations are listed in Table 1. Partial-duplex substrates were prepared as follows; one of the two oligonucleotides (10 pmol) in a partial-duplex DNA substrate was 5′-labeled with 3.3 pmol of [γ-32P] ATP and polynucleotide kinase according to the manufacturer's protocol. The labeled oligonucleotides were then annealed to the second oligonucleotide in 50 mM HEPES–NaOH/pH 7.5 and 200 mM NaCl, using a PCR machine (95°C: 5 min, 65°C: 30 min and −0.5°C/min to 25°C). For the preparation of flap-structured or nicked-duplex DNA substrates, one oligonucleotide was 5′-end labeled as described above and then annealed to the template and upstream oligonucleotides using a molar ratio of 1:2:4, respectively. To prepare DNA substrates containing two 5′-flaps (as shown in Figure 3), four-way junction (Figure 7) and replication fork-like substrates (fixed or partially movable double-strand three-way junction and regressed replication fork; shown in Figures 8A and 9), one of the oligonucleotides was labeled as described above and annealed with other three oligonucleotides at a molar ratio of 1:1:1:1. For the preparation of movable double-stranded three-way junction substrate (Figure 8B, C and D), two oligonucleotides 11 and 35 were first labeled and then annealed (1:1 ratio) separately to oligonucleotides 14 and 34, respectively. Subsequently, both partial-duplex DNAs were mixed together (1:1) and allowed to anneal for 12 h at 25°C. All annealed substrates were purified by 10% PAGE and the amount of recovered substrate was determined as previously described (14).Table 1.

Bottom Line: Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase.For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap.We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Center for DNA Replication and Genome Instability, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.

ABSTRACT
Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.

Show MeSH
Related in: MedlinePlus