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Pso2 (SNM1) is a DNA structure-specific endonuclease.

Tiefenbach T, Junop M - Nucleic Acids Res. (2011)

Bottom Line: Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast.In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates.Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.

ABSTRACT
Many types of DNA structures are generated in response to DNA damage, repair and recombination that require processing via specialized nucleases. DNA hairpins represent one such class of structures formed during V(D)J recombination, palindrome extrusion, DNA transposition and some types of double-strand breaks. Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast. Pso2 (SNM1A in mammals) belongs to a small group of proteins thought to function predominantly during interstrand crosslink (ICL) repair. In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates. Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease. This activity was further shown to be required in vivo for repair of chromosomal breaks harboring closed hairpin ends. These findings provide the first evidence that Pso2 may function outside ICL repair and open the possibility that Pso2 may function at least in part during ICL repair by processing DNA intermediates including DNA hairpins or hairpin-like structures.

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Pso2 possesses 5′ but not 3′-exonuclease activity and requires a 5′-phosphate for nucleotide removal. Pso2 (80 nM) was incubated with ssDNA (100 nM) labelled at the 5′ (A) or 3′-end (B). Purified pso2 H611A, a catalytic mutant (1 μM) was assayed with ssDNA (100 nM) labelled at the 5′ (C) or 3′-end (D). Lane 1 in each panel contains no Pso2. Reactions in lanes 2–10 were carried out for increasing time intervals: 0.25, 0.5, 0.75, 1, 2, 4, 8, 16 and 32 min, respectively. In panel (E) Pso2 (1.5 μM) was assayed using 3′-labelled 20 nt ssDNA (200 nM) containing a 5′-hydroxyl. Lane 1 is a negative control in the absence of Pso2. Reactions in lanes 2–8 were incubated for 0.5, 1, 2, 3, 4, 8 and 16 min, respectively. Panel (F) is identical to panel (E) except substrate used contains a 5′-phosphate. (G) EMSA reactions using increasing amounts of Pso2 were incubated with a 3′-labelled 12 nt single-stranded DNA substrate (100 nM) containing a phosphorothioate substitution between the first and second nucleotide as well as either a 5′-phosphate or a 5′-hydroxyl. Lane 1 and 5, no Pso2 added; lanes 2–4 contain 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively. Lanes 6–8, 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively.
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gkr1059-F1: Pso2 possesses 5′ but not 3′-exonuclease activity and requires a 5′-phosphate for nucleotide removal. Pso2 (80 nM) was incubated with ssDNA (100 nM) labelled at the 5′ (A) or 3′-end (B). Purified pso2 H611A, a catalytic mutant (1 μM) was assayed with ssDNA (100 nM) labelled at the 5′ (C) or 3′-end (D). Lane 1 in each panel contains no Pso2. Reactions in lanes 2–10 were carried out for increasing time intervals: 0.25, 0.5, 0.75, 1, 2, 4, 8, 16 and 32 min, respectively. In panel (E) Pso2 (1.5 μM) was assayed using 3′-labelled 20 nt ssDNA (200 nM) containing a 5′-hydroxyl. Lane 1 is a negative control in the absence of Pso2. Reactions in lanes 2–8 were incubated for 0.5, 1, 2, 3, 4, 8 and 16 min, respectively. Panel (F) is identical to panel (E) except substrate used contains a 5′-phosphate. (G) EMSA reactions using increasing amounts of Pso2 were incubated with a 3′-labelled 12 nt single-stranded DNA substrate (100 nM) containing a phosphorothioate substitution between the first and second nucleotide as well as either a 5′-phosphate or a 5′-hydroxyl. Lane 1 and 5, no Pso2 added; lanes 2–4 contain 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively. Lanes 6–8, 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively.

Mentions: Nuclease activity of highly purified Pso2 (Supplementary Figure S3) was analyzed using a variety of DNA substrates. In agreement with work by Li et al. (32), Pso2 was found to possess 5′- but not 3′-exonuclease activity (Figure 1). Our analysis further demonstrates that Pso2 is able to fully degrade the entire DNA substrate to a single mononucleotide indicating that Pso2 does not have a minimum DNA length requirement for activity.Figure 1.


Pso2 (SNM1) is a DNA structure-specific endonuclease.

Tiefenbach T, Junop M - Nucleic Acids Res. (2011)

Pso2 possesses 5′ but not 3′-exonuclease activity and requires a 5′-phosphate for nucleotide removal. Pso2 (80 nM) was incubated with ssDNA (100 nM) labelled at the 5′ (A) or 3′-end (B). Purified pso2 H611A, a catalytic mutant (1 μM) was assayed with ssDNA (100 nM) labelled at the 5′ (C) or 3′-end (D). Lane 1 in each panel contains no Pso2. Reactions in lanes 2–10 were carried out for increasing time intervals: 0.25, 0.5, 0.75, 1, 2, 4, 8, 16 and 32 min, respectively. In panel (E) Pso2 (1.5 μM) was assayed using 3′-labelled 20 nt ssDNA (200 nM) containing a 5′-hydroxyl. Lane 1 is a negative control in the absence of Pso2. Reactions in lanes 2–8 were incubated for 0.5, 1, 2, 3, 4, 8 and 16 min, respectively. Panel (F) is identical to panel (E) except substrate used contains a 5′-phosphate. (G) EMSA reactions using increasing amounts of Pso2 were incubated with a 3′-labelled 12 nt single-stranded DNA substrate (100 nM) containing a phosphorothioate substitution between the first and second nucleotide as well as either a 5′-phosphate or a 5′-hydroxyl. Lane 1 and 5, no Pso2 added; lanes 2–4 contain 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively. Lanes 6–8, 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr1059-F1: Pso2 possesses 5′ but not 3′-exonuclease activity and requires a 5′-phosphate for nucleotide removal. Pso2 (80 nM) was incubated with ssDNA (100 nM) labelled at the 5′ (A) or 3′-end (B). Purified pso2 H611A, a catalytic mutant (1 μM) was assayed with ssDNA (100 nM) labelled at the 5′ (C) or 3′-end (D). Lane 1 in each panel contains no Pso2. Reactions in lanes 2–10 were carried out for increasing time intervals: 0.25, 0.5, 0.75, 1, 2, 4, 8, 16 and 32 min, respectively. In panel (E) Pso2 (1.5 μM) was assayed using 3′-labelled 20 nt ssDNA (200 nM) containing a 5′-hydroxyl. Lane 1 is a negative control in the absence of Pso2. Reactions in lanes 2–8 were incubated for 0.5, 1, 2, 3, 4, 8 and 16 min, respectively. Panel (F) is identical to panel (E) except substrate used contains a 5′-phosphate. (G) EMSA reactions using increasing amounts of Pso2 were incubated with a 3′-labelled 12 nt single-stranded DNA substrate (100 nM) containing a phosphorothioate substitution between the first and second nucleotide as well as either a 5′-phosphate or a 5′-hydroxyl. Lane 1 and 5, no Pso2 added; lanes 2–4 contain 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively. Lanes 6–8, 5′-hydroxyl substrate with final Pso2 concentrations of 50, 100 and 200 nM, respectively.
Mentions: Nuclease activity of highly purified Pso2 (Supplementary Figure S3) was analyzed using a variety of DNA substrates. In agreement with work by Li et al. (32), Pso2 was found to possess 5′- but not 3′-exonuclease activity (Figure 1). Our analysis further demonstrates that Pso2 is able to fully degrade the entire DNA substrate to a single mononucleotide indicating that Pso2 does not have a minimum DNA length requirement for activity.Figure 1.

Bottom Line: Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast.In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates.Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.

ABSTRACT
Many types of DNA structures are generated in response to DNA damage, repair and recombination that require processing via specialized nucleases. DNA hairpins represent one such class of structures formed during V(D)J recombination, palindrome extrusion, DNA transposition and some types of double-strand breaks. Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast. Pso2 (SNM1A in mammals) belongs to a small group of proteins thought to function predominantly during interstrand crosslink (ICL) repair. In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates. Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease. This activity was further shown to be required in vivo for repair of chromosomal breaks harboring closed hairpin ends. These findings provide the first evidence that Pso2 may function outside ICL repair and open the possibility that Pso2 may function at least in part during ICL repair by processing DNA intermediates including DNA hairpins or hairpin-like structures.

Show MeSH
Related in: MedlinePlus