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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 opens DNA hairpin structures. DNA substrates containing either a single 3-nt hairpin (A) or covalently closed double 3-nt hairpin (B) were interrogated with T7 ds- and RecJf ss-exonuclease activities to verify structural integrity of each substrate. In (A) T7 ds-exonuclease is able to degrade the hairpin to half its original size, while RecJf had no effect since there is no ssDNA present in this substrate. Pso2 opens the hairpin on the 3′-side of the apex. In (B) neither RecJf or T7 exonuclease activities were able to degrade the fully closed double hairpin structure. Pso2 opened the hairpin structures and subsequently degraded the substrate using its 5′-exonuclease activity. (C) Pso2 WT and pso2 H611A (5 μM) were assayed with linear pUC19 plasmid. pso2 H611A shows no exonuclease activity. (D) Pso2 WT and pso2 H611A (1 μM) were incubated with a completely closed hairpin substrate comprised of 20-bp duplex, two 3-nt DNA hairpins at each end and an internal label.
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gkr1059-F2: Pso2 opens DNA hairpin structures. DNA substrates containing either a single 3-nt hairpin (A) or covalently closed double 3-nt hairpin (B) were interrogated with T7 ds- and RecJf ss-exonuclease activities to verify structural integrity of each substrate. In (A) T7 ds-exonuclease is able to degrade the hairpin to half its original size, while RecJf had no effect since there is no ssDNA present in this substrate. Pso2 opens the hairpin on the 3′-side of the apex. In (B) neither RecJf or T7 exonuclease activities were able to degrade the fully closed double hairpin structure. Pso2 opened the hairpin structures and subsequently degraded the substrate using its 5′-exonuclease activity. (C) Pso2 WT and pso2 H611A (5 μM) were assayed with linear pUC19 plasmid. pso2 H611A shows no exonuclease activity. (D) Pso2 WT and pso2 H611A (1 μM) were incubated with a completely closed hairpin substrate comprised of 20-bp duplex, two 3-nt DNA hairpins at each end and an internal label.

Mentions: Results obtained during the analysis of Pso2 exonuclease activity (Supplementary Figure S4F) suggested that Pso2 might be able to open DNA hairpin structures. To verify this activity, we analyzed the ability of Pso2 to process a doubly hairpinned substrate containing no nicks. The integrity of the fully closed hairpin structure was confirmed by treatment with both ssDNA and dsDNA exonucleases (RecJf and T7) (Figure 2B). As expected, the hairpin substrate was completely resistant to both exonuclease activities. In Figure 2B, the ability of Pso2 to open a DNA hairpin structure even when there were no nicks present in the substrate is demonstrated. A Pso2 mutant lacking exonuclease activity (H611A) failed to open hairpin structures (Figure 1C and D) indicating that Pso2 makes use of a single active site for both endo- and exonuclease functions. Taken together with the finding that endonuclease activity does not occur on linear ssDNA or dsDNA substrates (Figure 1, Supplementary Figure S4D and E), this data strongly suggests that Pso2 is a DNA hairpin, structure-specific endonuclease. Additionally, it also demonstrates that Pso2 is capable of opening fully paired DNA hairpins in substrates lacking a nick. This is in direct contrast to Mre11, an enzyme well characterized for its hairpin processing ability, which has severely reduced endonuclease activity towards fully paired hairpins and appears to function with Sae2 on DNA hairpins containing a single strand nick (8).Figure 2.


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

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

Pso2 opens DNA hairpin structures. DNA substrates containing either a single 3-nt hairpin (A) or covalently closed double 3-nt hairpin (B) were interrogated with T7 ds- and RecJf ss-exonuclease activities to verify structural integrity of each substrate. In (A) T7 ds-exonuclease is able to degrade the hairpin to half its original size, while RecJf had no effect since there is no ssDNA present in this substrate. Pso2 opens the hairpin on the 3′-side of the apex. In (B) neither RecJf or T7 exonuclease activities were able to degrade the fully closed double hairpin structure. Pso2 opened the hairpin structures and subsequently degraded the substrate using its 5′-exonuclease activity. (C) Pso2 WT and pso2 H611A (5 μM) were assayed with linear pUC19 plasmid. pso2 H611A shows no exonuclease activity. (D) Pso2 WT and pso2 H611A (1 μM) were incubated with a completely closed hairpin substrate comprised of 20-bp duplex, two 3-nt DNA hairpins at each end and an internal label.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr1059-F2: Pso2 opens DNA hairpin structures. DNA substrates containing either a single 3-nt hairpin (A) or covalently closed double 3-nt hairpin (B) were interrogated with T7 ds- and RecJf ss-exonuclease activities to verify structural integrity of each substrate. In (A) T7 ds-exonuclease is able to degrade the hairpin to half its original size, while RecJf had no effect since there is no ssDNA present in this substrate. Pso2 opens the hairpin on the 3′-side of the apex. In (B) neither RecJf or T7 exonuclease activities were able to degrade the fully closed double hairpin structure. Pso2 opened the hairpin structures and subsequently degraded the substrate using its 5′-exonuclease activity. (C) Pso2 WT and pso2 H611A (5 μM) were assayed with linear pUC19 plasmid. pso2 H611A shows no exonuclease activity. (D) Pso2 WT and pso2 H611A (1 μM) were incubated with a completely closed hairpin substrate comprised of 20-bp duplex, two 3-nt DNA hairpins at each end and an internal label.
Mentions: Results obtained during the analysis of Pso2 exonuclease activity (Supplementary Figure S4F) suggested that Pso2 might be able to open DNA hairpin structures. To verify this activity, we analyzed the ability of Pso2 to process a doubly hairpinned substrate containing no nicks. The integrity of the fully closed hairpin structure was confirmed by treatment with both ssDNA and dsDNA exonucleases (RecJf and T7) (Figure 2B). As expected, the hairpin substrate was completely resistant to both exonuclease activities. In Figure 2B, the ability of Pso2 to open a DNA hairpin structure even when there were no nicks present in the substrate is demonstrated. A Pso2 mutant lacking exonuclease activity (H611A) failed to open hairpin structures (Figure 1C and D) indicating that Pso2 makes use of a single active site for both endo- and exonuclease functions. Taken together with the finding that endonuclease activity does not occur on linear ssDNA or dsDNA substrates (Figure 1, Supplementary Figure S4D and E), this data strongly suggests that Pso2 is a DNA hairpin, structure-specific endonuclease. Additionally, it also demonstrates that Pso2 is capable of opening fully paired DNA hairpins in substrates lacking a nick. This is in direct contrast to Mre11, an enzyme well characterized for its hairpin processing ability, which has severely reduced endonuclease activity towards fully paired hairpins and appears to function with Sae2 on DNA hairpins containing a single strand nick (8).Figure 2.

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