The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases

Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn2+ on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3′-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11–13 nt away in the top strand and 9–10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5′-CN11–13↓N9–10G-3′/3′-GN9–10↓N11–13C-5′, with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells

PKCδ Clustering at the Leading Edge and Mediating Growth Factor-Enhanced, but not ECM-Initiated, Dermal Fibroblast Migration

We have previously shown that the immobilized extracellular matrices (ECMs) initiate cell migration and soluble growth factors (GFs) further enhance ECM-initiated cell migration. GFs alone cannot initiate cell migration. To further investigate the specificity of the two signaling mechanisms, we focused on the protein kinase C (PKC) family genes in primary human dermal fibroblasts (DFs). We here show that platelet-derived growth factor-BB (PDGF-BB) strongly stimulates membrane translocation and leading edge clustering of protein kinase Cδ (PKCδ). In contrast, attachment to collagen matrix alone does not cause the translocation. Although the kinase function of PKCδ is dispensable for initial membrane translocation, it is critical for its sustained presence at the cells's leading edge. Blockade of endogenous PKCδ signaling with dominant-negative kinase-defective PKC (PKCδ-KD) or PKCδ-small interfering RNA (siRNA) completely inhibited PDGF-BB-stimulated DF migration. In contrast, neither PKCδ-KD nor PKCδ-siRNA affected collagen-induced initiation of DF migration. Overexpression of a constitutively activated PKCδ (PKCδ-R144/145A) partially mimics the effect of PDGF-BB. However, PKCδ-KD, PKCδ-siRNA, or PKCδ-R144/145A does not affect PDGF-BB-stimulated activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, or c-Jun N-terminal kinase. Instead, inhibition of PKCδ blocks PDGF-BB-stimulated activation of signal transducer and activator of transcription 3 (Stat3). This study unveiled the specificity of PKCδ in the control of DF migration

Alteration of Sequence Specificity of the Type IIS Restriction Endonuclease BtsI

The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type

Rolling circle reverse transcription enables high fidelity nanopore sequencing of small RNA.

Small RNAs (sRNAs) are an important group of non-coding RNAs that have great potential as diagnostic and prognostic biomarkers for treatment of a wide variety of diseases. The portability and affordability of nanopore sequencing technology makes it ideal for point of care and low resource settings. Currently sRNAs can't be reliably sequenced on the nanopore platform due to the short size of sRNAs and high error rate of the nanopore sequencer. Here, we developed a highly efficient nanopore-based sequencing strategy for sRNAs (SR-Cat-Seq) in which sRNAs are ligated to an adapter, circularized, and undergo rolling circle reverse transcription to generate concatemeric cDNA. After sequencing, the resulting tandem repeat sequences within the individual cDNA can be aligned to generate highly accurate consensus sequences. We compared our sequencing strategy with other sRNA sequencing methods on a short-read sequencing platform and demonstrated that SR-Cat-Seq can obtain low bias and highly accurate sRNA transcriptomes. Therefore, our method could enable nanopore sequencing for sRNA-based diagnostics and other applications

The Fidelity Index provides a systematic

quantitation of star activity of DNA restriction endonuclease

Run-off sequencing to determine the actual cutting site of BtsI-1.

<p>A: pUC19 (0.6 µg) was digested by BtsI-1(∼1.8 pmol) and subjected to run-off sequencing with two flanking primers for both directions. The drop in the peak reflects where the polymerase runs off the template at the nicked sites. The terminal “a” is automatically added to the end by the polymerase during sequencing. B: The sequencing data were aligned with pUC19 sequence. The nicking sites are indicated by arrows on both strands.</p

Specific activity of BtsI(B:R119K) mutant on pUC19.

<p>BtsIB(R119K) was partially purified, 2-fold serially diluted and mixed with constant amount of BtsIA (∼43 pmol) to digest pUC19 (0.6 µg) in NEB4 at 55°C for 1 hour. Lane 1: 1 kb DNA ladder.</p

Purification of BtsIA and BtsIB(R119A) with IMPACT system.

<p>1 µl (lane 2), 2 µl (lane 3) and 4 µl (lane 4) of eluted BtsIA protein (A) or BtsIB(R119A) (B) were resolved by SDS-PAGE and stained with Coomassie Blue. Lane 1: protein ladder (NEB #P7711S)</p