Structural and biophysical characterization of murine rif1 C terminus reveals high specificity for DNA cruciform structures.

Mammalian Rif1 is a key regulator of DNA replication timing, double-stranded DNA break repair, and replication fork restart. Dissecting the molecular functions of Rif1 is essential to understand how it regulates such diverse processes. However, Rif1 is a large protein that lacks well defined functional domains and is predicted to be largely intrinsically disordered; these features have hampered recombinant expression of Rif1 and subsequent functional characterization. Here we applied ESPRIT (expression of soluble proteins by random incremental truncation), an in vitro evolution-like approach, to identify high yielding soluble fragments encompassing conserved regions I and II (CRI and CRII) at the C-terminal region of murine Rif1. NMR analysis showed CRI to be intrinsically disordered, whereas CRII is partially folded. CRII binds cruciform DNA with high selectivity and micromolar affinity and thus represents a functional DNA binding domain. Mutational analysis revealed an α-helical region of CRII to be important for cruciform DNA binding and identified critical residues. Thus, we present the first structural study of the mammalian Rif1, identifying a domain that directly links its function to DNA binding. The high specificity of Rif1 for cruciform structures is significant given the role of this key protein in regulating origin firing and DNA repair.

The recognition domain of the methyl-specific endonuclease McrBC flips out 5-methylcytosine.

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.

MuA-based Molecular Indexing for Rare Mutation Detection by Next-Generation Sequencing.

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.

4-[N-(substituted 4-pyrimidinyl)amino]benzenesulfonamides as inhibitors of carbonic anhydrase isozymes I, II, VII, and XIII.

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.

Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family.

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.

Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1).

Rif1 is a conserved protein that plays essential roles in orchestrating DNA replication timing, controlling nuclear architecture, telomere length and DNA repair. However, the relationship between these different roles, as well as the molecular basis of Rif1 function is still unclear. The association of Rif1 with insoluble nuclear lamina has thus far hampered exhaustive characterization of the associated protein complexes. We devised a protocol that overcomes this problem, and were thus able to discover a number of novel Rif1 interactors, involved in chromatin metabolism and phosphorylation. Among them, we focus here on PP1. Data from different systems have suggested that Rif1-PP1 interaction is conserved and has important biological roles. Using mutagenesis, NMR, isothermal calorimetry and surface plasmon resonance we demonstrate that Rif1 is a high-affinity PP1 adaptor, able to out-compete the well-established PP1-inhibitor I2 in vitro. Our conclusions have important implications for understanding Rif1 diverse roles and the relationship between the biological processes controlled by Rif1.

In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique.

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.

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution.

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-A 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.