No abstract

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

The Thermus aualcus DNA methyltransferase M'Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of -CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethlonlne shows a/P folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide deft suitable to accommodate the DNA substrate. The N-terminal domain Is dominated by a nine-stranded 3-sheet; it contains the two conserved segments typical for N-methyltranserases which form a pocket for cofactor binding. The C-terminal domain is formed by four small 1-sheets and a-helices. The three-dimensional folding of M'Taq I is similar to that of the cytosine-speciflc Hha I methyltransferase, where the large 1-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.DNA-methyltransferases (MTases) are a family of enzymes that occur in nearly all living organisms. They catalyze the transfer of-CH3 from the cofactor S-adenosyl-L-methionine (AdoMet) to cytosine C5 (C-MTases) or cytosine N4 or adenine N6 (N-MTases) in di-to octanucleotide target sequences of double-stranded DNA (1). In bacteria, all three types of MTases are found and implicated in the protection of DNA from their own restriction endonucleases and in mismatch repair (2). In eukaryotes only C-MTases have been observed so far; they are involved in cell differentiation, genome imprinting, mutagenesis, and regulation of gene expression (3).The C-MTases are a homogeneous class of molecules with three-dimensional structures probably similar to the structure described recently for the M-Hha I enzyme from Haemophilus haemolyticus (4). This is because their amino acid sequences show sequential arrangement of 10 conserved segments (I to X) from the N to the C terminus (5); segments I (DXFXGXG, with X = any amino acid) and IV (FPCQ) are implicated in binding of AdoMet, and the cysteine in IV is involved in the transfer of -CH3. In contrast, the N-MTases show only two of the conserved segments (6). They correspond to segments I and IV in the C-MTases, namely I (DXFXGXG), which can degenerate so much that only one glycine is retained, and II (DPPY), where aspartate can be replaced by asparagine or seine, and tyrosine by phenylalanine. Because these two segments can occur in reversed order-i.e., one or the other N-terminal (7)-the N-MTases are a more heterogeneous class of molecules. When the amino acid sequences of only those N-MTases that recognize TNNA (N = any nucleotide) are compared, an additional segment III is found (8). It spans 38 amino acids, has no equivalent in C-MTases, and occurs sequentially-i.e., I, II, III. The mechanism of methyl transfer is different in C-and N-MTases. In the former the conserved cysteine SH in segment IV attacks C6 of cytosine to form a covalent intermediate with resonance-stabilized carbanionic ...

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonucleaseDNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.DNA recognition sequences ofsite-specific proteins are embedded in a large background of nonspecific DNA. Nevertheless, such proteins locate their recognition sites by kinetically efficient processes, with reported apparent second-order association rate constants approaching or exceeding those expected for diffusion-limited collision of macromolecules (1). These surprising findings led to the suggestion that the effective target size for specific DNA-protein interaction might be much larger than the size of the recognition sequence (1, 2). In such models the protein is envisioned to interact with both specific and nonspecific DNA sequences, with interactions of the latter type being on the major kinetic path by which the protein locates its recognition site. Thus, random collisions between protein and DNA would initially favor formation of nonspecific complexes, which could then be converted to site-specific complexes by a facilitated diffusion mechanism in which the protein might, for example, "slide" along the polynucleotide via a random walk until the recognition sequence is encountered.Although theoretical treatments (2-4) have indicated that such a mechanism can account for the kinetic efficiency of sitespecific proteins, direct experimental support has been limited (1,(5)(6)(7)(8). A minimal test ofthis type ofmechanism requires proof of interaction of the protein in question with nonspecific sequences and demonstration that such interactions are involved in the path by which the protein locates and leaves its recognition site. In this paper we demonstrate that DNA sequences external to the recognition site markedly enhance the rate at which endodeoxyribonuclease EcoRI (EC 3.1.23.13) locates and leaves its recognition site. The external sequences, however, are without effect on the intrinsic equilibrium constant governing specific interactions. These findings, are in accord with a facilitated diffusion mechanism for specific DNA site location by this enzyme. A preliminary account of this work has been presented (5).MATERIALS AND METHODS Enzymes. EcoRI endonuclease was the homogeneou...

Vent DNA polymerase normally discriminates strongly against incorporation of ribonucleotides, 3'-deoxyribonucleotides (such as cordycepin) and 2',3'-dideoxyribonucleotides. To explore the basis for this discrimination we have generated a family of variants with point mutations of residues in conserved Regions II and III and assayed incorporation of nucleo-tides with modified sugars by these variants, all of which were created in an exonuclease-deficient form of the enzyme. A Y412V variant incorporates ribonucleotides at least 200-fold more efficiently than the wild-type enzyme, consistent with Y412 acting as a 'steric gate' to specifically exclude ribonucleotides. The most striking variants tested involved changes to A488, a residue predicted to be facing away from the nucleotide binding site. The pattern of relaxed specificity at this position roughly correlates with the size of the substituted amino acid sidechain and affects a variety of modified nucleotide sugars.