Detection of low-frequency mutations in cancer genomes or other heterogeneous cell populations requires high-fidelity sequencing. Molecular barcoding is one of the key technologies that enables the differentiation of true mutations from errors, which can be caused by sequencing or library preparation processes. However, current approaches where barcodes are introduced via primer extension or adaptor ligation do not utilize the full power of barcoding, due to complicated library preparation workflows and biases. Here we demonstrate the remarkable tolerance of MuA transposase to the presence of multiple replacements in transposon sequence, and explore this unique feature to engineer the MuA transposome complex with randomised nucleotides in 12 transposon positions, which can be introduced as a barcode into the target molecule after transposition event. We applied the approach of Unique MuA-based Molecular Indexing (UMAMI) to assess the power of rare mutation detection by shortgun sequencing on the Illumina platform. Our results show that UMAMI allows detection of rare mutations readily and reliably, and in this paper we report error rate values for the number of thermophilic DNA polymerases measured by using UMAMI.

Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are disrupted during denaturation step (94-96°C) in the first circles of PCR. Released plasmid DNA that encodes target polymerase and the thermophilic enzyme complement the emulsified PCR reaction mixture and start polymerase gene amplification. To be able to select for mezophilic enzymes we have employed multiple freezing-thawing cycles of emulsion as a bacterial cell wall disruption step instead of high temperature incubation. Subsequently WGA like plasmid DNA amplification could be performed by phi29 DNA polymerase applying different selection pressure conditions (temperature, buffer composition, modified dNTP, time, etc.). In our case the library of random phi29 DNA polymerase mutants was subjected to seven selection rounds of isothermal CSR (iCSR). After the selection polymerase variant containing the most frequent mutations was constructed and characterized. The mutant phi29 DNA polymerase can perform WGA at elevated temperatures (40-42°C), generate two to five times more of DNA amplification products, and has significantly increased half-life at 30 and 40°C, both in the presence or the absence of DNA substrate.

DNA cytosine methylation is a widespread epigenetic mark. Biological effects of DNA methylation are mediated by the proteins that preferentially bind to 5-methylcytosine (5mC) in different sequence contexts. Until now two different structural mechanisms have been established for 5mC recognition in eukaryotes; however, it is still unknown how discrimination of the 5mC modification is achieved in prokaryotes. Here we report the crystal structure of the N-terminal DNA-binding domain (McrB-N) of the methyl-specific endonuclease McrBC from Escherichia coli. The McrB-N protein shows a novel DNA-binding fold adapted for 5mC-recognition. In the McrB-N structure in complex with methylated DNA, the 5mC base is flipped out from the DNA duplex and positioned within a binding pocket. Base flipping elegantly explains why McrBC system restricts only T4-even phages impaired in glycosylation [Luria, S. E. and Human, M. L. (1952) A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol., 64, 557-569]: flipped out 5-hydroxymethylcytosine is accommodated in the binding pocket but there is no room for the glycosylated base. The mechanism for 5mC recognition employed by McrB-N is highly reminiscent of that for eukaryotic SRA domains, despite the differences in their protein folds.

A series of 4-[N-(substituted 4-pyrimidinyl)amino]benzenesulfonamides were designed and synthesised. Their binding potencies as inhibitors of selected recombinant human carbonic anhydrase (hCA) isozymes I, II, VII, and XIII were measured using isothermal titration calorimetry and the thermal shift assay. To determine the structural features of inhibitor binding, the crystal structures of several compounds in complex with hCA II were determined. Several compounds exhibited selectivity towards isozymes I, II, and XIII, and some were potent inhibitors of hCA VII.

II tipo restrikcijos endonukleazės atpažįsta specifines DNR sekas ir kerpa DNR siose sekose arba salia jų. BpuJI, atpažįstanti 5’-CCCGT seką, skiriasi nuo kitų fermentų tuo, kad jos kirpimo vieta yra labai variabili. Cia parodoma, kad BpuJI yra dimeras, sudarytas is dviejų monomerų, kurie turi po du atskirus domenus. BpuJI N domenas atpažįsta taikinį kaip monomeras, o C-domenas pasižymi nukleaziniu aktyvumu ir dimerizuojasi. Apo-fermento nukleazinis aktyvumas yra nuslopintas. N-domenams atpažinus taikinį, aktyvuojamas C-domenas, kuris perkerpa DNR salia taikinio. Be to, aktyvuotas C-domenas yra nespecifinė nukleazė, linkusi nukirpti ~3 nt nuo buko dvigrandės DNR galo. Taigi, BpuJI DNR karpymo pobūdis yra labai sudėtingas. Bioinformatinė analizė ir kryptinga mutagenezė parodė, kad BpuJI C-domenas turi PD-(D/E)XK struktūrine sanklodą ir yra panasus į archėjų Holidėjaus jungtis karpancias nukleazes. Issprendus 1,3 A skiriamosios gebos BpuJI N-domeno/DNR komplekso erdvinė struktūrą, paaiskėjo, kad sį domeną sudaro du „sparnuotą“ spiralė-linkis-spiralė motyvą turintys subdomenai. BpuJI taikinį atpažįsta aminorūgstys, esancios N-rankoje ir abiejų spiralė-linkis-spiralė motyvų atpažinimo spiralėse. BpuJI N-domenas yra labiausiai panasus į Nt.BspD6I nukleaze, kerpancią vieną DNR grandine. Nt.BspD6I/DNR komplekso struktūros modelis rodo, kad Nt.BspD6I ir BpuJI taikinį atpažįstantys struktūriniai elementai yra panasūs.%%%%Type II restriction endonucleases recognize specific DNA sequences and cleave DNA at fixed positions within or close to this sequence. BpuJI recognizes the 5’-CCCGT sequence, but in contrast to other enzymes its cleavage site is very variable. This study shows that BpuJI is a dimer in solution and consists of two separate domains. The N-domain binds to the target sequence as a monomer, while the C-domain is responsible for nuclease activity and dimerization. The nuclease activity is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the N-domains. The activated C-domain cleaves DNA near the target site. In addition, it possesses an end-directed nuclease activity and preferentially cuts ~3 nt from the 3’ terminus. This leads to a very complicated pattern of DNA cleavage. Bioinformatics and mutational analysis revealed that the BpuJI C-domain harbours a PD (D/E)XK active site and is structurally related to archaeal Holliday junction resolvases. The crystal structure of the BpuJI N-domain bound to cognate DNA was solved at 1.3 A resolution. It revealed two winged-helix subdomains, D1 and D2. The recognition of the target sequence is achieved the amino acid residues located on both the HTH motifs and an N-terminal arm. The BpuJI DNA recognition domain is most similar to the nicking endonuclease Nt.BspD6I. The modelling suggests that Nt.BspD6I could share the specificity-determining regions with BpuJI.

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein–DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix–turn–helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.

Human carbonic anhydrases are potential drug targets for a number of diseases.One of the novel applications is to use some of their isozymes as anti-cancer drug targets.Structure-thermodynamic property relations of novel hCA thiadiazole class inhibitors with a triple-ring system bound to hCAII will be discussed.Structures of several inhibitors are solved to atomic resolution using X-ray diffraction of hCAII-inhibitor complex crystals.The structural data are correlated with the isothermal titration calorimetry measurements.The calorimetric data together with the structures provide insight into the structural base of the tight and selective hCA inhibitor binding.

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5'-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3'-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.