Technical note: correcting for shear strain in an oscillatory squeeze flow rheometer

Konigsberg, David,Nicholson, Timothy M.,Halley, Peter J.,Ahn, Kyung Hyun Springer-Verlag 2014 Rheologica Acta: an international journal of rheol Vol.53 No.2

Hydrogen-Bonding Capability of a Templating Difluorotoluene Nucleotide Residue in an RB69 DNA Polymerase Ternary Complex

Xia, Shuangluo,Konigsberg, William H.,Wang, Jimin American Chemical Society 2011 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.133 No.26

<P>Results obtained using 2,4-difluorotoluene nucleobase (dF) as a nonpolar thymine isostere by Kool and colleagues challenged the Watson–Crick dogma that hydrogen bonds between complementary bases are an absolute requirement for accurate DNA replication. Here, we report crystal structure of an RB69 DNA polymerase L561A/S565G/Y567A triple mutant ternary complex with a templating dF opposite dTTP at 1.8 Å-resolution. In this structure, direct hydrogen bonds were observed between: (i) dF and the incoming dTTP, (ii) dF and residue G568 of the polymerase, and (iii) dF and ordered water molecules surrounding the nascent base pair. Therefore, this structure provides evidence that a templating dF can form novel hydrogen bonds with the incoming dTTP and with the enzyme that differ from those formed with a templating dT.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/2011/jacsat.2011.133.issue-26/ja2021735/production/images/medium/ja-2011-021735_0002.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ja2021735'>ACS Electronic Supporting Info</A></P>

DNA Mismatch Synthesis Complexes Provide Insights into Base Selectivity of a B Family DNA Polymerase

Xia, Shuangluo,Wang, Jimin,Konigsberg, William H. American Chemical Society 2013 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.135 No.1

<P>Current hypotheses that attempt to rationalize the high degree of base selectivity exhibited by replicative DNA polymerases (pols) concur that ternary complexes formed with incorrect dNTPs are destabilized. Knowing what accounts for this destabilization is likely to be the key to understanding base discrimination. To address this issue, we have determined crystal structures of ternary complexes with all 12 mismatches using an engineered RB69 pol quadruple mutant (qm, L415A/L561A/S565G/Y567A) that enabled us to capture nascent mispaired dNTPs. These structures show that mismatches in the nascent base-pair binding pocket (NBP) of the qm pol differ markedly from mismatches embedded in binary pol–DNA complexes. Surprisingly, only 3 of 12 mismatches clash with the NBP when they are modeled into the wild-type (wt) pol. The remaining can fit into a wt pol ternary complex but deviate from normal Watson–Crick base-pairs. Repositioning of the templating nucleotide residue and the enlarged NBP in qm ternary complex play important roles in accommodating incorrect incoming dNTPs. From these structures, we propose additional reasons as to why incorrect dNTPs are incorporated so inefficiently by wt RB69 pol: (i) steric clashes with side chains in the NBP after Fingers closing; (ii) weak interactions or large gaps between the incoming dNTP and the templating base; and (iii) burying a protonated base in the hydrophobic environment of the NBP. All of these possibilities would be expected to destabilize the closed ternary complex so that incorporation of incorrect dNTP would be a rare event.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/2013/jacsat.2013.135.issue-1/ja3079048/production/images/medium/ja-2012-079048_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ja3079048'>ACS Electronic Supporting Info</A></P>

Structural Basis for DifferentialInsertion Kineticsof dNMPs Opposite a Difluorotoluene Nucleotide Residue

Xia, Shuangluo,Eom, Soo Hyun,Konigsberg, William H.,Wang, Jimin American ChemicalSociety 2012 Biochemistry Vol.51 No.7

<P>We have recently challenged the widely held view that2,4-difluorotoluene(dF) is a nonpolar isosteric analogue of the nucleotide dT, incapableof forming hydrogen bonds (HBs). To gain a further understanding forthe kinetic preference that favors dAMP insertion opposite a templatingdF, a result that mirrors the base selectivity that favors dAMP insertionopposite dT by RB69 DNA polymerase (RB69pol), we determined presteady-statekinetic parameters for incorporation of four dNMPs opposite dF byRB69pol and solved the structures of corresponding ternary complexes.We observed that both the F2 and F4 substituent of dF in these structuresserve as HB acceptors forming HBs either directly with dTTP and dGTPor indirectly with dATP and dCTP via ordered water molecules. We havedefined the shape and chemical features of each dF/dNTP pair in theRB69pol active site without the corresponding phosphodiester-linkageconstraints of dF/dNs when they are embedded in isolated DNA duplexes.These features can explain the kinetic preferences exhibited by thetemplating dF when the nucleotide incorporation is catalyzed by wildtype RB69pol or its mutants. We further show that the shapes of thedNTP/dF nascent base pair differ markedly from the corresponding dNTP/dTin the pol active site and that these differences have a profoundeffect on their incorporation efficiencies.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2012/bichaw.2012.51.issue-7/bi2016487/production/images/medium/bi-2011-016487_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/bi2016487'>ACS Electronic Supporting Info</A></P>

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

Xia, Shuangluo,Christian, Thomas D.,Wang, Jimin,Konigsberg, William H. American Chemical Society 2012 Biochemistry Vol.51 No.21

<P>Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2′-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10<SUP>2</SUP>–10<SUP>3</SUP>-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10<SUP>2</SUP>–10<SUP>3</SUP>-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position <I>n</I> – 2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2012/bichaw.2012.51.issue-21/bi300416z/production/images/medium/bi-2012-00416z_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/bi300416z'>ACS Electronic Supporting Info</A></P>

Using a Fluorescent Cytosine Analogue tC^o To Probe the Effect of the Y567 to Ala Substitution on the Preinsertion Steps of dNMP Incorporation by RB69 DNA Polymerase

Xia, Shuangluo,Beckman, Jeff,Wang, Jimin,Konigsberg, William H. American Chemical Society 2012 Biochemistry Vol.51 No.22

<P>Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC<SUP>o</SUP>), to identify preinsertion step(s) altered by NBP flexibility. When tC<SUP>o</SUP> is the templating base in a wild-type (wt) RB69pol ternary complex, its fluorescence is quenched only in the presence of dGTP. However, with the RB69pol Y567A mutant, the fluorescence of tC<SUP>o</SUP> is also quenched in the presence of dATP. We determined the crystal structure of the dATP/tC<SUP>o</SUP>-containing ternary complex of the RB69pol Y567A mutant at 1.9 Å resolution and found that the incoming dATP formed two hydrogen bonds with an imino-tautomerized form of tC<SUP>o</SUP>. Stabilization of the dATP/tC<SUP>o</SUP> base pair involved movement of the tC<SUP>o</SUP> backbone sugar into the DNA minor groove and required tilting of the tC<SUP>o</SUP> tricyclic ring to prevent a steric clash with L561. This structure, together with the pre-steady-state kinetic parameters and dNTP binding affinity, estimated from equilibrium fluorescence titrations, suggested that the flexibility of the NBP, provided by the Y567 to Ala substitution, led to a more favorable forward isomerization step resulting in an increase in dNTP binding affinity.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2012/bichaw.2012.51.issue-22/bi300241m/production/images/medium/bi-2012-00241m_0005.gif'></P>

Contribution of Partial Charge Interactions and Base Stacking to the Efficiency of Primer Extension at and beyond Abasic Sites in DNA

Xia, Shuangluo,Vashishtha, Ashwani,Bulkley, David,Eom, Soo Hyun,Wang, Jimin,Konigsberg, William H. American Chemical Society 2012 Biochemistry Vol.51 No.24

<P>During DNA synthesis, base stacking and Watson–Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB’s interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge–charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB’s surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2012/bichaw.2012.51.issue-24/bi300296q/production/images/medium/bi-2012-00296q_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/bi300296q'>ACS Electronic Supporting Info</A></P>

Insights into Base Selectivity from the 1.8 Å Resolution Structure of an RB69 DNA Polymerase Ternary Complex

Wang, Mina,Xia, Shuangluo,Blaha, Gregor,Steitz, Thomas A.,Konigsberg, William H.,Wang, Jimin American Chemical Society 2011 Biochemistry Vol.50 No.4

<P/><P>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) <I>Cell 105</I>, 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.</P>

Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3′-terminal phosphate and 5′-OH

Englert, Markus,Xia, Shuangluo,Okada, Chiaki,Nakamura, Akiyoshi,Tanavde, Ved,Yao, Min,Eom, Soo Hyun,Konigsberg, William H.,Sö,ll, Dieter,Wang, Jimin Proceedings of the National Academy of Sciences 2012 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.109 No.38

<P>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<SUP>2+</SUP>-dependent reaction. Here, we report two crystal structures of <I>Pyrococcus horikoshii</I> RNA-splicing ligase RtcB in complex with Mn<SUP>2+</SUP> alone (RtcB/ Mn<SUP>2+</SUP>) and together with a covalently bound GMP (RtcB-GMP/Mn<SUP>2+</SUP>). The RtcB/ Mn<SUP>2+</SUP> structure (at 1.6 Å resolution) shows two Mn<SUP>2+</SUP> 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<SUP>2+</SUP> 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<SUP>2+</SUP> 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>i</I>) guanylylation of the enzyme, (<I>ii</I>) guanylyl-transfer to the RNA substrate, and (<I>iii</I>) 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<SUP>2+</SUP> 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.</P>