Randomized DNA libraries construction tool: a new 3-bp 'frequent cutter' TthHB27I/sinefungin endonuclease with chemically-induced specificity.
ABSTRACT: BACKGROUND:Acoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~?3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN. RESULTS:In this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5'-CAARCA-3' is converted into a combined 3.2-3.0-bp 'site' or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed. CONCLUSIONS:In the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA.
Project description:BACKGROUND: Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases). RESULTS: We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) - an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5'-GACCGA-3' [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation. CONCLUSIONS: In the presence of SIN/DMSO, TaqII REase is transformed from cleaving every 4096 bp on average to cleaving every 58 bp. TaqII SIN/DMSO thus extends the palette of available REase prototype specificities. This phenomenon, employed under partial digestion conditions, was applied to quasi-random DNA fragmentation. Further applications include high sensitivity probe generation and metagenomic DNA amplification.
Project description:Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4-8 bp long, with very few exceptions cleaving DNA more frequently. TsoI is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R?=?A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus-family exhibit 'affinity star' activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue-sinefungin (SIN). Here, we define a novel type of inherently built-in 'star' activity, exemplified by TsoI. The TsoI 'star' activity cannot be described under the definition of the classic 'star' activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The TsoI SCS comprises several degenerated variants of the cognate site. Although the efficiency of TsoI SCS cleavage is lower in comparison to the cognate TsoI recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The TsoI/SAC REase may serve as a novel tool for DNA manipulation.
Project description:BACKGROUND: We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI. RESULTS: TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases. CONCLUSIONS: TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.
Project description:BACKGROUND: In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI. RESULTS: TsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined. CONCLUSIONS: Previously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism's thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.
Project description:Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca(2+), SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca(2+), substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca(2+) and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.
Project description:BACKGROUND: We developed a web-based, prognostic tool for extremity and trunk wall soft tissue sarcoma to predict 10-year sarcoma-specific survival. External validation was performed. METHODS: Patients referred during 1987-2002 to Helsinki University Central Hospital are included. External validation was obtained from the Lund University Hospital register. Cox proportional hazards models were fitted with the Helsinki data. The previously described model (SIN) includes size, necrosis, and vascular invasion. The extended model (SAM) includes the SIN factors and in addition depth, location, grade, and size on a continuous scale. Models were statistically compared according to accuracy (area under the ROC curve=AUC) of 10-year sarcoma-specific survival prediction. RESULTS: The AUC of the SAM model in 10-year survival prediction in the Helsinki patient series was 0.81 as compared with 0.74 for the SIN model (P=0.0007). The corresponding AUCs in the external validation series were 0.77 for the SAM model and 0.73 for the SIN model (P=0.03). A web-based calculator for the SAM model is available at http://www.prognomics.org/sam. CONCLUSION: Addition of grade, depth, and location as well as tumour size on a continuous scale significantly improved the accuracy of the prognostic model when compared with a model that includes only size, necrosis, and vascular invasion.
Project description:Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3'-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex.
Project description:Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.
Project description:Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.
Project description:Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5'-CC/WGG-3' (where W = A/T, and '/' marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5'-CCWGG-3' sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5'-CCWGG-3' sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.