Proteins encoded by the DpnII restriction gene cassette

Campa, Adela G. de la; Kale, P. G.; Springhorn, Sylvia S.; Lacks, Sanford A.

doi:10.1016/0022-2836(87)90024-6

Cited by 80 publications

(48 citation statements)

References 31 publications

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“…ScrFI (Davis et al, 1993), BcnI (Vilkaitis et al, 2002), DpnII (de la Campa et al, 1987), LlaDCHI (Moineau et al, 1995), MboI (Ueno et al, 1993)] possess two MTases with the same specificity can be linked to a possible sensitivity of one or both enzymes to the Mg 2+ . In such circumstances, to ensure proper methylation of genomic DNA, it would make sense to employ in this process two enzymes with the same methylating activity.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

2003

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The gene encoding the LlaCI methyltransferase (M.LlaCI) from Lactococcus lactis subsp. cremoris W15 was overexpressed in Escherichia coli. The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of M r 31 300±1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 59-AAGCTT-39. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 59 end of the specific sequence to N 6 -methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzae Rd) and EcoVIII (Escherichia coli E1585-68) restriction-modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among N 6 -adenine b-class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg 2+ for phosphodiester bond cleavage. Mg 2+ was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg 2+ may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed. INTRODUCTIONLactic acid bacteria (LAB) are probiotic micro-organisms that have been used for millennia in the processing of dairy products. It was shown recently that dairy establishments in Britain can be dated back to 5000 years BC (Copley et al., 2003). Manufacturing of food such as cheese and yogurt, but also sauerkraut and dill pickles, as well as the existence of some regional cuisine, is heavily dependent on lactic fermentation. LAB and food processed by them therefore have armies of admirers, but on the other side, these bacteria have also powerful enemies. Their enemies are not people who hate buttermilk, but virulent phages whose existence relies on propagation within bacterial cells. To combat phages, LAB have developed multiple molecular strategies.These measures can be divided into four groups: (i) inhibition of phage adsorption, (ii) blockage of phage DNA injection, (iii) abortive infection mechanisms, and (iv) restriction of phage DNA (Forde & Fitzgerald, 1999;Coffey & Ross, 2002). These defence mechanisms are often located on plasmids. That feature can facilitate their horizontal spread among LAB and when acquired affords them protection against phage invasion (Allison & Klaenhammer, 1998). Among the defence mechanisms listed above, restriction-modification (R-M) systems form the most diverse group. Based on their molecular structure and cofactor requireme...

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Section: Discussionmentioning

confidence: 99%

Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

2003

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“…In our experiments, we used ~20 nM (~10 molecules per µm 3 or lower, see Methods section and Supplementary Fig. S1) type II restriction enzymes with an approximate size of d E ≈ 5 nm 16,19,33 , which are known to form stable homodimers in solutions 16 Fluorescence images of seq5 dsDnA forming a homogeneous sAm on an ultraflat gold surface, respectively, before and after 1-h incubation in a solution with BamHI restriction enzyme. Line profiles c1 and d1 show that the reaction does not take place within dsDnA molecules if the alkylthiol square is not generated.…”

Section: Resultsmentioning

confidence: 99%

Two-dimensional enzyme diffusion in laterally confined DNA monolayers

et al. 2011

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Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DnA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDnAs from the topmatrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top-and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon.

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“…Procedures used for the growth and transformation of E. coli and for hyperexpression of Dpn II system enzymes have been reported (11). The latter depended on induction of T7 RNA polymerase in BL21(DE3) hosts (15).…”

Section: Methodsmentioning

confidence: 99%

“…All three enzymes recognize 5'-GATC-3' sequences in DNA, but DpnA appeared to be less constrained than the others in its specificity (3,11). In this work, an improved procedure for purifying large amounts of the DpnA methylase was devised.…”

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confidence: 99%