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Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.

Horton JR, Wang H, Mabuchi MY, Zhang X, Roberts RJ, Zheng Y, Wilson GG, Cheng X - Nucleic Acids Res. (2014)

Bottom Line: We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically.MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits.Such interchange of function might explain how other complex multimeric restriction enzymes act.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.

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MspJI mutagenesis. (a) SDS-PAGE showing the deletion mutants used for activity. (b) Activities of MspJI WT and deletion mutants performed as 4-fold serial titrations (lanes 1–4). Approximately equal amount of purified enzymes was added into the starting point of each serial dilution. Based on the cleavage activity showed on the gel, we estimate that the decrease in activity for all the deletion mutants is greater than 64-fold. (c) Activity of K173F mutant.
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Figure 4: MspJI mutagenesis. (a) SDS-PAGE showing the deletion mutants used for activity. (b) Activities of MspJI WT and deletion mutants performed as 4-fold serial titrations (lanes 1–4). Approximately equal amount of purified enzymes was added into the starting point of each serial dilution. Based on the cleavage activity showed on the gel, we estimate that the decrease in activity for all the deletion mutants is greater than 64-fold. (c) Activity of K173F mutant.

Mentions: The properties of a number of point mutants of MspJI (8), and of a related enzyme, AspBHI (9), were reported earlier. Additional mutations, described below, were constructed during this study. Loop-45 (residues 90–103), between strands β4 and β5, includes Ser90 and Trp92 at the beginning of the loop, and Trp101 and Asp103 at the end, which perform critical functions in the 5mC-recognition pocket (Figure 3c and d). This loop adopts a somewhat different conformation according to whether the pocket is occupied (subunit C), partially occupied (subunit D) or empty (subunits A and B). Deletion derivatives were constructed that lacked residues 95 and 96 (mutant Δ95–96) or residues 95 through 98 (Δ95–98) (Figure 4a). Both mutant proteins were almost inactive, confirming the importance of the loop. The residual activity they displayed resulted mainly in nicked substrate plasmid DNA (Figure 4b).


Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.

Horton JR, Wang H, Mabuchi MY, Zhang X, Roberts RJ, Zheng Y, Wilson GG, Cheng X - Nucleic Acids Res. (2014)

MspJI mutagenesis. (a) SDS-PAGE showing the deletion mutants used for activity. (b) Activities of MspJI WT and deletion mutants performed as 4-fold serial titrations (lanes 1–4). Approximately equal amount of purified enzymes was added into the starting point of each serial dilution. Based on the cleavage activity showed on the gel, we estimate that the decrease in activity for all the deletion mutants is greater than 64-fold. (c) Activity of K173F mutant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231741&req=5

Figure 4: MspJI mutagenesis. (a) SDS-PAGE showing the deletion mutants used for activity. (b) Activities of MspJI WT and deletion mutants performed as 4-fold serial titrations (lanes 1–4). Approximately equal amount of purified enzymes was added into the starting point of each serial dilution. Based on the cleavage activity showed on the gel, we estimate that the decrease in activity for all the deletion mutants is greater than 64-fold. (c) Activity of K173F mutant.
Mentions: The properties of a number of point mutants of MspJI (8), and of a related enzyme, AspBHI (9), were reported earlier. Additional mutations, described below, were constructed during this study. Loop-45 (residues 90–103), between strands β4 and β5, includes Ser90 and Trp92 at the beginning of the loop, and Trp101 and Asp103 at the end, which perform critical functions in the 5mC-recognition pocket (Figure 3c and d). This loop adopts a somewhat different conformation according to whether the pocket is occupied (subunit C), partially occupied (subunit D) or empty (subunits A and B). Deletion derivatives were constructed that lacked residues 95 and 96 (mutant Δ95–96) or residues 95 through 98 (Δ95–98) (Figure 4a). Both mutant proteins were almost inactive, confirming the importance of the loop. The residual activity they displayed resulted mainly in nicked substrate plasmid DNA (Figure 4b).

Bottom Line: We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically.MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits.Such interchange of function might explain how other complex multimeric restriction enzymes act.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.

Show MeSH