Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity

Abstract

Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4–8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.

Common Copy Number Variation Detection From Multiple Sequenced Samples

“…7 labeled with “CNVnator” and “proposed” show that the daughter (NA12878) shares more CNVs with her mother (NA12892) than her father (NA12891), suggesting that the daughter is ge-nomically closer to her mother than her father. This is consistent with the results of Magi et al [54], and ours reported before [55]. …”

Multiple‐cumulative probabilities used to cluster and visualize transcriptomes

“…Moreover, these clustering results were displayed on t‐SNE‐MCP‐O, t‐SNE‐N and t‐SNE‐C maps (Fig. 7), where t‐SNE‐C was t‐SNE of the centered gene points 7. From Fig.…”

“…However, hierarchical clustering was likely to cause loose gene neighbors. That is, two neighbors that were generated by the hierarchical clustering were not really the nearest and second‐closest neighbors of genes 7.…”

“…Moreover, clustering number of data sets mainly came from the corresponding references. In detail, data set 2 had been divided into eight clusters by Euclidean distance 7, data set 3 into 25 clusters by PoissonC and TransChisq 12, 15, data set 4 into six and 10 clusters by Euclidean distance 7, and data set 5 into eight clusters by Euclidean distance 14.…”

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