Decreased Km to dNTPs is an essential M-MuLV reverse transcriptase adoption required to perform efficient cDNA synthesis in One-Step RT-PCR assay.

Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display technique for in vitro evolution of the Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT) that would be able to perform efficient full-length cDNA synthesis in PCR buffer optimized for Thermus aquaticus DNA polymerase. The most frequent mutations found in a selected library were analyzed. Aside from the mutations, which switch off RNase H activity of RT and are beneficial for the full-length cDNA synthesis, we have identified several mutations in the active center of the enzyme (Q221R and V223A/M), which result in 4-5-fold decrease of Km for dNTPs (<0.2 mM). The selected mutations are in surprising agreement with the natural evolution process because they transformed the active center from the oncoretroviral M-MuLV RT-type to the lenitiviral enzyme-type. We believe that this was the major and essential phenotypic adjustment required to perform fast and efficient cDNA synthesis in PCR buffer at 0.2-mM concentration of each dNTP.

Structure-based redesign of the catalytic/metal binding site of Cfr10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues.

According to the crystal structure of Cfr10I restriction endonuclease the acidic residues D134, E71 and E204 are clustered together and presumably chelate metal ion(s) at the active site. Indeed, investigation of the DNA cleavage properties of substitutional mutants of Cfr10I D134A, E71Q, E71A and E204Q reveals that D134, E71 and E204 residues are essential for cleavage activity, supporting their active site function. Structural comparison indicates that the D134 residue of Cfr10I spatially overlaps with aspartate residues D91 and D74, from the invariant active site motifs 90PDX19EAK and 73PDX15DIK of EcoRI and EcoRV, respectively. However, structural studies in conjunction with mutational analyses suggest that the sequence motif 133PDX55KX13E corresponds to the active site of Cfr10I, but differs from canonical active site motifs of EcoRI and EcoRV. According to the crystal structure of Cfr10I the serine S188 residue from the 188SVK sequence motif is a spatial equivalent of the acidic residue from the (E/D)XK-part of the active site motif, which is conserved between EcoRI and EcoRV. Site-directed mutagenesis experiments of Cfr10I, however, revealed that the S188 was not so important for catalysis while the E204 residue located 2.8 A away indeed was essential for cleavage, suggesting that the glutamate E204 rather than the S188 residue contributes to the metal binding site in Cfr10I. In addition, model-building studies suggest that mutual interchange of the E204 and S188 residues should lead only to minor positional differences of the carboxylate residues of glutamate side-chains. The double mutant S188E/E204S was therefore prepared by site-directed mutagenesis where the active site motif 133PDX55KX13E of Cfr10I was changed to a canonical motif 133PDX53EVK, which is similar to that of EcoRI and EcoRV. Interestingly, the double mutant S188E/E204S of Cfr10I with redesigned active site structure, exhibited 10% of Wt cleavage activity in a gamma DNA cleavage assay. Thus, structure guided redesign of the catalytic/metal binding site of Cfr10I, provides novel experimental evidence to suggest that spatial rather than sequence conservation plays the dominant role in the formation of restriction enzyme active sites.

The Cfr10I restriction enzyme is functional as a tetramer.

It is thought that most of the type II restriction endonucleases interact with DNA as homodimers. Cfr10I is a typical type II restriction enzyme that recognises the 5'-Pu decreases CCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in isolation. The only SfiI restriction enzyme that recognises the long interrupted recognition sequence 5'-GGCCNNNNNGGCC has been previously reported to operate as a tetramer however, its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine, and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that, in contrast to the wild-type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mutant is < 0.1% of that of the wild-type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I.

Ca2+-ions stimulate DNA binding specificity of Cfr10I restriction enzyme.

The Cfr10I restriction enzyme recognizes the degenerate hexanucleotide sequence 5'-Pu/CCGGPy-3' and cleaves it as indicated. DNA binding studies of Cfr10I endonuclease were performed using gel mobility shift assay. Analysis of Cfr10I binding to DNA revealed that in the absence of metal ions Cfr10I binds to DNA containing or lacking the recognition sequence with similar low affinity. Addition of Ca2+ to the binding buffer stimulated manifestation of DNA binding specificity of Cfr10I.