Bacteriophage RB69 DNA polymerase (RB69 pol) has served as a model for investigating how B family polymerases achieve a high level of fidelity during DNA replication. We report here the structure of an RB69 pol ternary complex at 1.8 Å resolution, extending the resolution from our previously reported structure at 2.6 Å [Franklin, M. C., et al. (2001) Cell 105, 657−667]. In the structure presented here, a network of five highly ordered, buried water molecules can be seen to interact with the N3 and O2 atoms in the minor groove of the DNA duplex. This structure reveals how the formation of the closed ternary complex eliminates two ordered water molecules, which are responsible for a kink in helix P in the apo structure. In addition, three pairs of polar−nonpolar interactions have been observed between (i) the Cα hydrogen of G568 and the N3 atom of the dG templating base, (ii) the O5′ and C5 atoms of the incoming dCTP, and (iii) the OH group of S565 and the aromatic face of the dG templating base. These interactions are optimized in the dehydrated environment that envelops Watson−Crick nascent base pairs and serve to enhance base selectivity in wild-type RB69 pol.

The RtcB protein has recently been identified as a 3′-phosphate RNA ligase that directly joins an RNA strand ending with a 2′,3′-cyclic phosphate to the 5′-hydroxyl group of another RNA strand in a GTP/Mn 2+ -dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn 2+ alone (RtcB/ Mn 2+) and together with a covalently bound GMP (RtcB-GMP/Mn 2+ ). The RtcB/ Mn 2+ structure (at 1.6 Å resolution) shows two Mn 2+ ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn 2+ complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn 2+ ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn 2+ active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3′-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5′-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.RNA repair | tRNA splicing | two-metal-ion catalysis R NA ligases join two RNA strands whose ends are produced by specific RNases in many biological processes during tRNA processing/splicing, antiphage, or unfolded protein response (1-3). Most widely studied are the 5′-Phosphate (5′-P) RNA ligases (4-6) that specifically catalyze the nucleophilic attack of a free 3′-hydroxyl on an activated 5′-P. However, these ligases cannot directly join two RNA strands ending with a 2′,3′-cyclic phosphate (RNA>p) and with a 5′-hydroxyl group. Before ligation, these strands require the action of a polynucleotide kinase (forming a 5′-P) and a phosphoesterase (generating a free 3′-OH) (6). The joining mechanism of 5′-P polynucleotide ligases involves a 3′-hydroxyl and an activated 5′-P end for internucleotide phosphodiester bond formation (7,8). The 5′-P activation enzymes include group I and II self-splicing introns, the spliceosomal apparatus, and DNA/RNA polymerases, as well as other nucleotidyl transferases (9-11). Similarly, the tRNA His guanylyltransferase ligates the 3′-OH of GTP to the 5′-terminus of tRNA through its adenylylation-activated 5′-P; this gives the appearance of reverse polarity of nucleotide addition relative to normal RNA polymerases (12).The 3′-Phosphate (3′-P) RNA ligase activities were identified three decades ago; they use a 3′-P as a donor for joining two RNAs without involvement of phosphorylation of the 5′-hydroxyl or dephosphorylation of the 2′,3′-cyclic phosphate (1...

We have captured a pre-insertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3’ hydroxyl group at the terminus of an extendable primer (ptO3’) and a non-hydrolyzable 2’-deoxyuridine 5’-α,β-substituted trisphosphate, dUpXpp, where X is either NH or CH2, opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca2+ and other four structures with catalytically competent Mn2+ or Mg2+. These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3’ close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3’ and the non-bridging Sp oxygen of the dNTP’s α-phosphate. The coordination bond length of metal-ion A as well as its ionic radius determines how close ptO3’ can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα enabling pyrophosphorolysis as well as assisting in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3’ occupies the vertex of a distorted metalion A coordination octahedron. When ptO3’ is placed into the vertex of a non-distorted, idealized metalion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.

SUMMARYThe plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 Å X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well-defined target DNA called oriT. The active site metal is observed near Tyr 25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism. KeywordsMobA; X-ray structure; DNA binding; drug resistance; relaxases Resistance to antibiotics by bacterial pathogens is a major health concern throughout the world. Bacteria acquire resistance to antibiotics most commonly by the conjugative transfer of plasmids and transposable elements. There are two classes of plasmids involved in conjugative transfer. One of these consists of large (greater than 30 Kb) self-transmissible elements 1 and the other smaller (~5-10 Kb) mobilizable plasmids 2 3 . Both groups encode the proteins required to process the DNA for transfer, but only the self-transmissible plasmids encode as well the complex, type IV secretion system (T4SS) required to transport the DNA into a new cell. The mobilizable plasmids use a T4SS that is generally provided by a co-resident, selftransmissible plasmid 4 , but can also take advantage of type IV systems involved in the transport of effector proteins active in pathogenesis 5 . An important characteristic of T4SS is that it can transport both protein and DNA across species barriers 6, 7 and even into cells of different kingdoms 8; 9 . In addition, some groups of plasmids are maintained in a large numberCorrespondence to: Jon D. Robertus. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of different bacterial species 10 . These two characteristics have together resulted ...