SsoII-like DNA-methyltransferase Ecl18kI: Interaction between regulatory and methylating functions

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Δ(72–379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyl-transferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.     

Iterative database searches demonstrate that glycoside hydrolase families 27, 31, 36 and 66 share a common evolutionary origin with family 13

A catalytic sequence motif PDX10-30(E/D)XK is found in many restriction enzymes. On the basis of sequence similarities and mapping of the conserved residues to the crystal structure of NgoMIV we suggest that residues D160, K182, R186, R188 and E195 contribute to the catalytic/DNA binding site of the Ecl18kI restriction endonuclease. Mutational analysis confirms the functional significance of the conserved residues of Ecl18kI. Therefore, we conclude that the active site motif 159VDX21KX12E of Ecl18kI differs from the canonical PDX10-30(E/D)XK motif characteristic for most of the restriction enzymes. Moreover, we propose that two subfamilies of endonucleases Ecl18kI/PspGI/EcoRII and Cfr10I/Bse634I/NgoMIV, specific, respectively, for CCNGG/CCWGG and RCCGGY/GCCGGC sites, share conserved active site architecture and DNA binding elements.     

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.     

DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer–dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.     

Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease

Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5'-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5'-overhangs. Here, we report the crystal structure of the Ecl18kI-DNA complex at 1.7 A resolution and compare it with the known structure of the NgoMIV-DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson-Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.     

How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets

Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This ‘double check’ mechanism explains their extraordinary specificity for an A/T pair in the flipping position.     

A novel gene, ecl1(+), extends the chronological lifespan in fission yeast.

We have identified a novel gene from Schizosaccharomyces pombe that we have named ecl1(+) (extender of the chronological lifespan). When ecl1(+) is provided on a high-copy number plasmid, it extends the viability of both the Deltasty1 MAP kinase mutant and the wild-type cells after entry into the stationary phase. ecl1(+) encodes an 80-amino acid polypeptide that had not been annotated in the current database. The ecl1(+)-mRNA increases transiently when the growth phase is changed from the log phase to the stationary phase. The Ecl1 protein is localized in the nucleus. Calorie restriction extends the chronological lifespan of wild-type and Deltaecl1 cells but not ecl1(+)-overproducing cells. The Deltapka1 mutant shows little, if any, additional extension of viability when Ecl1 is overproduced. The ste11(+) gene that is negatively controlled by Pka1 is up regulated when Ecl1 is overproduced. From these results we propose that the effect of Ecl1 overproduction may be mainly linked to and negatively affects the Pka1-dependent pathway.