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Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

Rockah-Shmuel L, Tawfik DS - Nucleic Acids Res. (2012)

Bottom Line: How then, do novel activities evolve?Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues.Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological chemistry, Weizmann Institute of Science, Hertzel St, Rehovot 76100, Israel.

ABSTRACT
DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

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Related in: MedlinePlus

Methylation of different target sites by wild-type M.HaeIII for detection of promiscuous activities. (A) End-point methylation activity assay of wild-type M.HaeIII of the original GGCC sequence, of promiscuous non-palindromic ‘star sites’ (AGCC, GGCT and GGCG) (11), and of the newly identified expanded palindromic sites: GGA/TCC (M.AvaII-like) and GGCGCC (M.NarI-like). These palindromic sites show similar specificity as the original site as indicated by the lower methylation of related ‘star’ sites (controls sites) ([E]0 = 2 µM; [DNA substrate] = 0.67 μM; [3H−SAM] = 0.2 μM; 20% glycerol; 5-h incubation time at 37°C). (B) Plasmid protection assay: The encoding plasmid of wild-type M.HaeIII was transformed to E. coli and was expressed without induction. The plasmid was subsequently extracted and treated with different restriction enzymes as noted. (C) Rates of H3-methyl incorporation were measured with the original DNA target (GGCC, right Y-axis), and with different promiscuous target sites (left Y-axis). Insert: the derived initial velocities with the different target sequences. ([E]0 = 0.2 µM; [DNA substrate] = 0.5 μM; [3H−SAM] = 0.2 μM; 20% glycerol at 37°C). UC, uncut plasmid.
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gks944-F1: Methylation of different target sites by wild-type M.HaeIII for detection of promiscuous activities. (A) End-point methylation activity assay of wild-type M.HaeIII of the original GGCC sequence, of promiscuous non-palindromic ‘star sites’ (AGCC, GGCT and GGCG) (11), and of the newly identified expanded palindromic sites: GGA/TCC (M.AvaII-like) and GGCGCC (M.NarI-like). These palindromic sites show similar specificity as the original site as indicated by the lower methylation of related ‘star’ sites (controls sites) ([E]0 = 2 µM; [DNA substrate] = 0.67 μM; [3H−SAM] = 0.2 μM; 20% glycerol; 5-h incubation time at 37°C). (B) Plasmid protection assay: The encoding plasmid of wild-type M.HaeIII was transformed to E. coli and was expressed without induction. The plasmid was subsequently extracted and treated with different restriction enzymes as noted. (C) Rates of H3-methyl incorporation were measured with the original DNA target (GGCC, right Y-axis), and with different promiscuous target sites (left Y-axis). Insert: the derived initial velocities with the different target sequences. ([E]0 = 0.2 µM; [DNA substrate] = 0.5 μM; [3H−SAM] = 0.2 μM; 20% glycerol at 37°C). UC, uncut plasmid.

Mentions: Plasmid DNA was extracted (from library pools or individual variants), treated with the restriction enzymes (10–20 U, 2 h at 37°C) and analyzed by gel electrophoresis. The same procedure was applied with genomic DNA extracted with the Sigma kit. DNA substrates for in vitro assays were prepared by primer extension (26-nt forward templates carrying a single restriction/methylation site, 12-nt biotinylated reverse primers, exo- Klenow fragment polymerase, NEB, 1 h, 37°C). The double stranded DNA products were analyzed on 4% agarose type XI gels (Sigma). A list of all DNA substrates is available in Supplementary Methods. ‘Time-dependent methylation assays’ were performed as described previously (19), using H3-labeled SAM (∼0.3 µM) and different enzyme and DNA substrate concentrations (10–700 nM; Supplementary Figure S5B). Aliquots taken at different time points were quenched and transferred to streptavidin-coated ScintiPlate wells (PerkinElmer) and H3 levels were determined using the Wallac MicroBeta TriLux counter (PerkinElmer). KMDNA and vmax values were derived by fitting initial rates to the Michaelis–Menten model using GraphPad Prism. Error ranges relate to the standard errors observed in two or more independent experiments. ‘End-point assays’ aimed at detecting weak and promiscuous activities (Figures 1 and 3), were performed as above but at saturating, rather than initial rate conditions, namely, using high enzyme concentration (2–4 µM) and long incubation times during which favored DNA substrates were completely methylated (0.5–5 h).Figure 1.


Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

Rockah-Shmuel L, Tawfik DS - Nucleic Acids Res. (2012)

Methylation of different target sites by wild-type M.HaeIII for detection of promiscuous activities. (A) End-point methylation activity assay of wild-type M.HaeIII of the original GGCC sequence, of promiscuous non-palindromic ‘star sites’ (AGCC, GGCT and GGCG) (11), and of the newly identified expanded palindromic sites: GGA/TCC (M.AvaII-like) and GGCGCC (M.NarI-like). These palindromic sites show similar specificity as the original site as indicated by the lower methylation of related ‘star’ sites (controls sites) ([E]0 = 2 µM; [DNA substrate] = 0.67 μM; [3H−SAM] = 0.2 μM; 20% glycerol; 5-h incubation time at 37°C). (B) Plasmid protection assay: The encoding plasmid of wild-type M.HaeIII was transformed to E. coli and was expressed without induction. The plasmid was subsequently extracted and treated with different restriction enzymes as noted. (C) Rates of H3-methyl incorporation were measured with the original DNA target (GGCC, right Y-axis), and with different promiscuous target sites (left Y-axis). Insert: the derived initial velocities with the different target sequences. ([E]0 = 0.2 µM; [DNA substrate] = 0.5 μM; [3H−SAM] = 0.2 μM; 20% glycerol at 37°C). UC, uncut plasmid.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526282&req=5

gks944-F1: Methylation of different target sites by wild-type M.HaeIII for detection of promiscuous activities. (A) End-point methylation activity assay of wild-type M.HaeIII of the original GGCC sequence, of promiscuous non-palindromic ‘star sites’ (AGCC, GGCT and GGCG) (11), and of the newly identified expanded palindromic sites: GGA/TCC (M.AvaII-like) and GGCGCC (M.NarI-like). These palindromic sites show similar specificity as the original site as indicated by the lower methylation of related ‘star’ sites (controls sites) ([E]0 = 2 µM; [DNA substrate] = 0.67 μM; [3H−SAM] = 0.2 μM; 20% glycerol; 5-h incubation time at 37°C). (B) Plasmid protection assay: The encoding plasmid of wild-type M.HaeIII was transformed to E. coli and was expressed without induction. The plasmid was subsequently extracted and treated with different restriction enzymes as noted. (C) Rates of H3-methyl incorporation were measured with the original DNA target (GGCC, right Y-axis), and with different promiscuous target sites (left Y-axis). Insert: the derived initial velocities with the different target sequences. ([E]0 = 0.2 µM; [DNA substrate] = 0.5 μM; [3H−SAM] = 0.2 μM; 20% glycerol at 37°C). UC, uncut plasmid.
Mentions: Plasmid DNA was extracted (from library pools or individual variants), treated with the restriction enzymes (10–20 U, 2 h at 37°C) and analyzed by gel electrophoresis. The same procedure was applied with genomic DNA extracted with the Sigma kit. DNA substrates for in vitro assays were prepared by primer extension (26-nt forward templates carrying a single restriction/methylation site, 12-nt biotinylated reverse primers, exo- Klenow fragment polymerase, NEB, 1 h, 37°C). The double stranded DNA products were analyzed on 4% agarose type XI gels (Sigma). A list of all DNA substrates is available in Supplementary Methods. ‘Time-dependent methylation assays’ were performed as described previously (19), using H3-labeled SAM (∼0.3 µM) and different enzyme and DNA substrate concentrations (10–700 nM; Supplementary Figure S5B). Aliquots taken at different time points were quenched and transferred to streptavidin-coated ScintiPlate wells (PerkinElmer) and H3 levels were determined using the Wallac MicroBeta TriLux counter (PerkinElmer). KMDNA and vmax values were derived by fitting initial rates to the Michaelis–Menten model using GraphPad Prism. Error ranges relate to the standard errors observed in two or more independent experiments. ‘End-point assays’ aimed at detecting weak and promiscuous activities (Figures 1 and 3), were performed as above but at saturating, rather than initial rate conditions, namely, using high enzyme concentration (2–4 µM) and long incubation times during which favored DNA substrates were completely methylated (0.5–5 h).Figure 1.

Bottom Line: How then, do novel activities evolve?Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues.Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological chemistry, Weizmann Institute of Science, Hertzel St, Rehovot 76100, Israel.

ABSTRACT
DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

Show MeSH
Related in: MedlinePlus