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

Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses

For a very long time, Type II restriction enzymes (REases) have been a paradigm of ORFans: proteins with no detectable similarity to each other and to any other protein in the database, despite common cellular and biochemical function. Crystallographic analyses published until January 2008 provided high-resolution structures for only 28 of 1637 Type II REase sequences available in the Restriction Enzyme database (REBASE). Among these structures, all but two possess catalytic domains with the common PD-(D/E)XK nuclease fold. Two structures are unrelated to the others: R.BfiI exhibits the phospholipase D (PLD) fold, while R.PabI has a new fold termed ‘half-pipe’. Thus far, bioinformatic studies supported by site-directed mutagenesis have extended the number of tentatively assigned REase folds to five (now including also GIY-YIG and HNH folds identified earlier in homing endonucleases) and provided structural predictions for dozens of REase sequences without experimentally solved structures. Here, we present a comprehensive study of all Type II REase sequences available in REBASE together with their homologs detectable in the nonredundant and environmental samples databases at the NCBI. We present the summary and critical evaluation of structural assignments and predictions reported earlier, new classification of all REase sequences into families, domain architecture analysis and new predictions of three-dimensional folds. Among 289 experimentally characterized (not putative) Type II REases, whose apparently full-length sequences are available in REBASE, we assign 199 (69%) to contain the PD-(D/E)XK domain. The HNH domain is the second most common, with 24 (8%) members. When putative REases are taken into account, the fraction of PD-(D/E)XK and HNH folds changes to 48% and 30%, respectively. Fifty-six characterized (and 521 predicted) REases remain unassigned to any of the five REase folds identified so far, and may exhibit new architectures. These enzymes are proposed as the most interesting targets for structure determination by high-resolution experimental methods. Our analysis provides the first comprehensive map of sequence-structure relationships among Type II REases and will help to focus the efforts of structural and functional genomics of this large and biotechnologically important class of enzymes

Nuclear architecture organized by Rif1 underpins the replication-timing program

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-{\AA} 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

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

International audienceMammalian 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

Domain architectures of () protein families sharing homology with the BpuJI nuclease domain identified in this study and () previously characterized Type III Res subunits and the McrBC system

<p><b>Copyright information:</b></p><p>Taken from "Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family"</p><p></p><p>Nucleic Acids Research 2007;35(7):2377-2389.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874659.</p><p>© 2007 The Author(s)</p> AHJR, the archaeal Holliday junction resolvase domain similar to the C-terminal catalytic domain of BpuJI; for domains of superfamily II helicases, SF-II, the closest homolog of known structure is indicated; HSP90N, the N-terminal domain of HSP90; McrB-N and McrB-C, respectively, the N-terminal DNA binding and the C-terminal GTPase domains of the McrB subunit of 5′-methylcytosine-specific restriction enzyme; DBD?, unclassified putative DNA binding domain; UPF0102, uncharacterized protein family with the predicted single-domain nuclease fold related to AHJRs; Region X and PD-(D/E)XK, endonuclease domains of Type III and McrC restriction enzymes, respectively. Each architecture in (A) is illustrated with a single representative and corresponds to the grouping in the alignment (Supplementary Figure 5B)

The 3′-end directed endonucleolytic activity of the isolated C-terminal domain of BpuJI

<p><b>Copyright information:</b></p><p>Taken from "Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family"</p><p></p><p>Nucleic Acids Research 2007;35(7):2377-2389.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874659.</p><p>© 2007 The Author(s)</p> () Cleavage of the PCR fragment by the C-terminal domain. Total of 1 nM of the PCR fragment, 5′-labelled in bottom strand, was incubated with 3 μM of the C-terminal domain at 4°C. Aliquots were removed at timed intervals (indicated above the relevant lanes) and analysed by electrophoresis through the standard sequencing gel. Lanes G, A, T, C are sequence ladders. () Cleavage of oligonucleotide duplexes by the C-terminal domain. Total of 2 nM of oligoduplex, 3′-labelled (upper gels) or 5′-labelled (lower gels) in top strand, was incubated with 100 nM of the C-terminal domain at 25°C. Aliquots were removed at timed intervals (indicated above the relevant lanes) and analysed by electrophoresis through denaturing 20% polyacrylamide. In cartoons below the gels the position of the label is indicated by a star, the arrows show the position of initial cleavage