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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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Related in: MedlinePlus

Kinetic analysis of Pso2 endonuclease activity. Kinetic analysis of Pso2 (80 nM) endonuclease activity was performed with a 3′-labelled DNA hairpin containing 9 bp of dsDNA, a single 3-nt hairpin and 5′-OH (100 nM). An 8-nt product is generated immediately following initiation of the reaction that is consistent with hairpin opening 2 nt from the apex on the 3′-side.
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gkr1059-F3: Kinetic analysis of Pso2 endonuclease activity. Kinetic analysis of Pso2 (80 nM) endonuclease activity was performed with a 3′-labelled DNA hairpin containing 9 bp of dsDNA, a single 3-nt hairpin and 5′-OH (100 nM). An 8-nt product is generated immediately following initiation of the reaction that is consistent with hairpin opening 2 nt from the apex on the 3′-side.

Mentions: To determine the exact location of DNA strand cleavage we performed kinetic analysis of Pso2 endonuclease activity using a 3′-32P-labelled hairpin substrate lacking a free 5′-PO4. The fidelity of this substrate was established via treatment with T7 and RecJf exonuclease activities. As expected, T7 exonuclease degraded the substrate to half its original size while RecJf had no effect (Figure 2A). This substrate is only susceptible to Pso2 exonuclease activity once the hairpin has been opened. Analysis of nuclease activity with this substrate (Figure 3) demonstrated the ability of Pso2 to open hairpin structures on the 3′-side of the apex and subsequently remove single nucleotides from the exposed 5′-PO4. The preferred site for cleavage was located two nucleotides away from the hairpin apex.Figure 3.


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

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

Kinetic analysis of Pso2 endonuclease activity. Kinetic analysis of Pso2 (80 nM) endonuclease activity was performed with a 3′-labelled DNA hairpin containing 9 bp of dsDNA, a single 3-nt hairpin and 5′-OH (100 nM). An 8-nt product is generated immediately following initiation of the reaction that is consistent with hairpin opening 2 nt from the apex on the 3′-side.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299998&req=5

gkr1059-F3: Kinetic analysis of Pso2 endonuclease activity. Kinetic analysis of Pso2 (80 nM) endonuclease activity was performed with a 3′-labelled DNA hairpin containing 9 bp of dsDNA, a single 3-nt hairpin and 5′-OH (100 nM). An 8-nt product is generated immediately following initiation of the reaction that is consistent with hairpin opening 2 nt from the apex on the 3′-side.
Mentions: To determine the exact location of DNA strand cleavage we performed kinetic analysis of Pso2 endonuclease activity using a 3′-32P-labelled hairpin substrate lacking a free 5′-PO4. The fidelity of this substrate was established via treatment with T7 and RecJf exonuclease activities. As expected, T7 exonuclease degraded the substrate to half its original size while RecJf had no effect (Figure 2A). This substrate is only susceptible to Pso2 exonuclease activity once the hairpin has been opened. Analysis of nuclease activity with this substrate (Figure 3) demonstrated the ability of Pso2 to open hairpin structures on the 3′-side of the apex and subsequently remove single nucleotides from the exposed 5′-PO4. The preferred site for cleavage was located two nucleotides away from the hairpin apex.Figure 3.

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