Abstract:LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, … Show more
“…The cleavage rate profile for pC1 shows the DNA nicking expected of a one-site DNA (Figure 1E). The rate is slower than that on either C2cat (Figure 1D) or pHT-12 (Figure 6F in (31)). The rate of cleavage of C1cat is faster than the corresponding pC1 (Figure 1E) but is similar to that of C2cat (Figure 1D).…”
To cleave DNA, the single polypeptide restriction–modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5′-CTnGAyG-3′ as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.
“…The cleavage rate profile for pC1 shows the DNA nicking expected of a one-site DNA (Figure 1E). The rate is slower than that on either C2cat (Figure 1D) or pHT-12 (Figure 6F in (31)). The rate of cleavage of C1cat is faster than the corresponding pC1 (Figure 1E) but is similar to that of C2cat (Figure 1D).…”
To cleave DNA, the single polypeptide restriction–modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5′-CTnGAyG-3′ as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.
“…The Type ISP RM systems such as LlaGI carry the gene fusion idea further than presented in this article as they have fused the HsdR to the MSFusion albeit using an MSFusion with only a single TRD (8). It would be of interest to create such a fusion from a classical Type I RM system such as EcoKI.…”
Section: Discussionmentioning
confidence: 99%
“…These systems often have multiple subunits, each expressing individual endonuclease, MTase or sequence recognition functions, but sometimes are found as a single polypeptide (SP) in which the subunits and their individual activities are fused together. Examples would be the SP enzymes RM.BpuSI (a Type IIG) (7) and LlaGI (a Type ISP with SP meaning single polypeptide) (8) or the Type IIB enzyme BcgI which fuses endonuclease and MTase function but keeps sequence recognition as a separate subunit (9,10). Fusions have been created artificially for a number of Type II RM systems and functionality has been maintained (11,12).…”
The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence.
“…The nuclease residues were mutated in pRSFLlaGI using the QuikChange mutagenesis protocol (see ‘Materials and Methods’ section). The wild type and mutant LlaGI enzymes were purified as described in the accompanying paper (Figure 2) (20); the mutants behaved as wild type during expression and purification (data not shown), with the exception of K102A which gave a reduced yield. Figure 2.Purification of LlaGI mutants.…”
Section: Resultsmentioning
confidence: 99%
“…DNA cleavage occurs at a random non-specific site and requires the interaction of at least two LlaGI enzymes bound at distant DNA sites (20). Long-range interaction is facilitated by a 1-D loop translocation mechanism (21).…”
Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction–modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
