Diet composition and feeding ecology of the naked goby Gobiosoma bosc (Gobiidae) from four western Atlantic estuaries.

The feeding ecology of the small-bodied benthic naked goby Gobiosoma bosc, a western Atlantic species that occurs in estuaries and other inshore habitats from Connecticut to Texas U.S.A., was investigated in a total of four estuaries spanning South Carolina, North Carolina, Maryland and New Jersey. Gut content analysis of 391 individuals revealed that G. bosc is a benthic microcarnivore that feeds primarily on polychaetes, gammarid amphipods and harpacticoid copepods. Diet composition varied with body size, tidal creek within an estuary and geographic region. Analyses of gut fullness suggest that G. bosc is a daytime visual predator and that nest and egg guarding during the reproductive season reduce foraging activity in mature males. Additionally, G. bosc infected with adult digenean parasites of the gut foraged more intensely than uninfected individuals, a relationship that was strongest for reproductively mature males. Regionally, significant variation in dietary breadth was documented and may reflect a foraging response to a decrease in prey diversity moving from estuaries of higher salinity and lower latitude to estuaries of lower salinity and higher latitude. These results contribute to an understanding of the life history of G. bosc and the role played by this common species in estuarine food webs.

Genetic population structure of spotted seatrout Cynoscion nebulosus along the south-eastern U.S.A.

Analyses of the genetic population structure of spotted seatrout Cynoscion nebulosus along the south-eastern U.S. coast using 13 microsatellites suggest significant population differentiation between fish in North Carolina (NC) compared with South Carolina (SC) and Georgia (GA), with New River, NC, serving as an area of integration between northern and southern C. nebulosus. Although there is a significant break in gene flow between these areas, the overall pattern throughout the sampling range represents a gradient in genetic diversification with the degree of geographic separation. Latitudinal distance and estuarine density appear to be main drivers in the genetic differentiation of C. nebulosus along the south-eastern U.S. coast. The isolation-by-distance gene-flow pattern creates fine-scale differences in the genetic composition of proximal estuaries and dictates that stocking must be confined to within 100 km of the location of broodstock collection in order to maintain the natural gradient of genetic variation along the south-eastern U.S. coast.

Study of the docking of competitive inhibitors at a model of tyrosinase active site: insights from joint broken-symmetry/Spin-Flip DFT computations and ELF topological analysis.

Following our previous study (Piquemal et al., New J. Chem., 2003, 27, 909), we present here a DFT study of the inhibition of the Tyrosinase enzyme. Broken-symmetry DFT computations are supplemented with Spin-Flip TD-DFT calculations, which, for the first time, are applied to such a dicopper enzyme. The chosen biomimetic model encompasses a dioxygen molecule, two Cu(II) cations, and six imidazole rings. The docking energy of a natural substrate, namely phenolate, together with those of several inhibitor and non-inhibitor compounds, are reported and show the ability of the model to rank the most potent inhibitors in agreement with experimental data. With respect to broken-symmetry calculations, the Spin-Flip TD-DFT approach reinforces the possibility for theory to point out potent inhibitors: the need for the deprotonation of the substrates, natural or inhibitors, is now clearly established. Moreover, Electron Localization Function (ELF) topological analysis computations are used to deeply track the particular electronic distribution of the Cu-O-Cu three-center bonds involved in the enzymatic Cu(2)O(2) metallic core (Piquemal and Pilmé, J. Mol. Struct.: Theochem, 2006, 77, 764). It is shown that such bonds exhibit very resilient out-of-plane density expansions that play a key role in docking interactions: their 3D-orientation could be the topological electronic signature of oxygen activation within such systems.

Simple Formulas for Improved Point-Charge Electrostatics in Classical Force Fields and Hybrid Quantum Mechanical/Molecular Mechanical Embedding.

We present a simple damping scheme for point-charge electrostatics that could be used directly in classical force fields. The approach acts at the charge (or monopole) level only and allows the inclusion of short-range electrostatic penetration effects at a very low cost. Results are compared with density functional theory Coulomb intermolecular interaction energies and with several other methods such as distributed multipoles, damped distributed multipoles, and transferable Hermite-Gaussian densities. Realistic trends in the interactions are observed for atom-centered Mertz-Kollman corrected point-charge distributions. The approach allows increasing the selectivity of parameters in the case of metal complexes. In addition, two QM/MM calculations are presented where the damping function is employed to include the MM atoms located at the QM/MM boundary. The first calculation corresponds to the gas-phase proton transfer of aspartic acid through water and the second is the first step of the reaction catalyzed by the 4-oxalocrotonate tautomerase (4OT) enzyme. First, improved agreement is observed when using the damping approach compared with the conventional excluded charge method or when including all charges in the calculation. Second, in the case of 4OT, the damped charge approach is in agreement with previous calculations, whereas including all charges gives a significantly higher energy barrier. In both cases, no reparameterization of the van der Waals part of the force field was performed.

Accumulation of methylmercury or polychlorinated biphenyls in in vitro models of rat neuronal tissue.

In vivo exposure levels for neurotoxicants are often reported in parts per million (ppm) concentration in tissue, whereas exposure levels in experiments utilizing in vitro models are most commonly reported in micromolar (muM) concentration in the exposure solution. The present experiments sought to determine whether or not in vitro solution concentration was an appropriate dose-metric for comparison to in vivo tissue levels for lipophilic compounds. To do so, the accumulation of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (A1254) or methylmercury (MeHg) was examined in three commonly utilized in vitro neuronal tissue models: nerve growth factor differentiated pheochromocytoma (PC12) cells, primary cultures of rat neocortical cells, and adult rat hippocampal slices. Tissues were exposed to A1254 (0.65 ppm) or to MeHg (0.0033-0.33 ppm) in serum-free media for 1 or 24 h. Total PCB or mercury accumulation was measured by dual column gas chromatography with electron capture detection or by cold vapor atomic absorption, respectively. PC12 cells accumulated 66.7 and 103.8 ppm PCBs after 1 and 24 h exposure to A1254. Neocortical neurons also accumulated significant concentrations of PCBs, but less so than PC12 cells. After 1 h exposure to 0.65 ppm A1254, slices contained 3.46 and 0.81 ppm PCBs when exposed in a static and perfused system, respectively. After 1 h exposure to 0.0033, 0.033, and 0.33 ppm MeHg, PC12 cells contained 0.3, 2.2, and 17.7 ppm mercury, respectively; after 24 h, PC12 cells contained 0.4, 2.8, and 21.9 ppm. Hippocampal slices accumulated 1.7 and 4.8 ppm mercury after 1 and 3 h exposure to 0.33 ppm MeHg. For comparison, mercury accumulation in rat fetal and pup brain tissue after maternal exposure [0, 0.1, 1.0, or 2.0 mg/kg/day MeHg from gestational day (GD) 6-15] ranged from 0.05 to 7.89 ppm in 0.1 mg/kg dose animals on postnatal day 10 and 2.0 mg/kg dose animals on GD16, respectively. These results demonstrate that accumulation of PCBs and MeHg in vitro is tissue-, time-, and concentration-dependent and indicates that tissue levels rather than exposure concentrations are a more appropriate metric for comparison of in vitro to in vivo effects.

Requirement for G proteins in insulin-like growth factor-I-induced growth of prostate cells.

Elevated levels of IGF-I in the circulation are associated with increased risk for the development of prostate cancer in men, and transgenic expression of human IGF-I in mouse epithelial prostate cells results in spontaneous prostate tumorigenesis. Little, however, is known about the mechanisms involved in the IGF-I-regulated growth of prostate cells. Here, we have demonstrated that treatment with IGF-I induces the activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and the growth of human prostate cells. Stimulation with IGF-I also promoted the tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Signal relay from IGF-I to ERK requires heterotrimeric G proteins and EGFR; inhibition of Gi/o protein activation by pertussis toxin, or EGFR by tyrphostin AG1478 obliterated the ability of IGF-I to promote ERK activation. Further, treatment with pertussis toxin inhibited the IGF-I-mediated prostate cell growth. These data demonstrated the requirement of heterotrimeric G proteins in IGF-I-regulated prostate cell growth and suggest the potential utility of the G proteins as effective drug targets to combat this common cancer.

An all-atom solution-equilibrated model for human extrinsic blood coagulation complex (sTF-VIIa-Xa): a protein-protein docking and molecular dynamics refinement study.

Tissue factor (TF)-bound factor (F)VIIa plays a critical role in activating FX, an event that rapidly results in blood coagulation. Despite recent advances in the structural information about soluble TF (sTF)-bound VIIa and Xa individually, the atomic details of the ternary complex are not known. As part of our long-term goal to provide a structural understanding of the extrinsic blood coagulation pathway, we built an all atom solution-equilibrated model of the human sTF-VIIa-Xa ternary complex using protein-protein docking and molecular dynamics (MD) simulations. The starting structural coordinates of sTF-VIIa and Xa were derived from dynamically equilibrated solution structures. Due to the flexible nature of the light-chain of the Xa molecule, a three-stage docking approach was employed in which SP (Arg195-Lys448)/EGF2 (Arg86-Arg139), EGF1 (Asp46-Thr85) and GLA (Ala1-Lys45) domains were docked in a sequential manner. The rigid-body docking approach of the FTDOCK method in conjunction with filtering based on biochemical knowledge from experimental site-specific mutagenesis studies provided the strategy. The best complex obtained from the docking experiments was further refined using MD simulations for 3 ns in explicit water. In addition to explaining most of the known experimental site-specific mutagenesis data pertaining to sTF-VIIa, our model also characterizes likely enzyme-binding exosites on FVIIa and Xa that may be involved in the ternary complex formation. According to the equilibrated model, the 140s loop of VIIa serves as the key recognition motif for complex formation. Stable interactions occur between the FVIIa 140s loop and the FXa -strand B2 region near the sodium-binding domain, the 160 s loop and the N-terminal activation loop regions. The helical-hydrophobic stack region that connects the GLA and EGF1 domains of VIIa and Xa appears to play a potential role in the membrane binding region of the ternary complex. The proposed model may serve as a reasonable structural basis for understanding the exosite-mediated substrate recognition of sTF-VIIa and to advance understanding of the TFPI-mediated regulatory pathway of the extrinsic blood coagulation cascade.

Gene selection for sample classification based on gene expression data: study of sensitivity to choice of parameters of the GA/KNN method.

MOTIVATION: We recently introduced a multivariate approach that selects a subset of predictive genes jointly for sample classification based on expression data. We tested the algorithm on colon and leukemia data sets. As an extension to our earlier work, we systematically examine the sensitivity, reproducibility and stability of gene selection/sample classification to the choice of parameters of the algorithm. METHODS: Our approach combines a Genetic Algorithm (GA) and the k-Nearest Neighbor (KNN) method to identify genes that can jointly discriminate between different classes of samples (e.g. normal versus tumor). The GA/KNN method is a stochastic supervised pattern recognition method. The genes identified are subsequently used to classify independent test set samples. RESULTS: The GA/KNN method is capable of selecting a subset of predictive genes from a large noisy data set for sample classification. It is a multivariate approach that can capture the correlated structure in the data. We find that for a given data set gene selection is highly repeatable in independent runs using the GA/KNN method. In general, however, gene selection may be less robust than classification. AVAILABILITY: The method is available at http://dir.niehs.nih.gov/microarray/datamining CONTACT: LI3@niehs.nih.gov

Expanding coincident signaling by PTEN through its inositol 1,3,4,5,6-pentakisphosphate 3-phosphatase activity.

PTEN, a tumor suppressor among the most commonly mutated proteins in human cancer, is recognized to be both a protein phosphatase and a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 3-phosphatase. Previous work [Maehama and Dixon, J. Biol. Chem. 273 (1998) 13375-13378] has led to a consensus that inositol phosphates are not physiologically relevant substrates for PTEN. In contrast, we demonstrate that PTEN is an active inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)) 3-phosphatase when expressed and purified from bacteria or HEK cells. Kinetic data indicate Ins(1,3,4,5,6)P(5) (K(m)=7.1 microM) and PtdIns(3,4,5)P(3) (K(m)=26 microM) compete for PTEN in vivo. Transient transfection of HEK cells with PTEN decreased Ins(1,3,4,5,6)P(5) levels. We discuss the physiological significance of these studies in relation to recent work showing that dephosphorylation of Ins(1,3,4,5,6)P(5) to inositol 1,4,5,6-tetrakisphosphate is a cell signaling event.

Modeling human zymogen factor IX.

Modern theoretical techniques are employed to provide complete three dimensional structure for the zymogen and activated forms of human coagulation factors IX and IXa. These structures are fully calcium bound and equilibrated in an electrically neutral aqueous environment. The relationship of structure to mutational data is examined. We find that a substantial relative orientational change of the catalytic domain occurs on activation. Also, we find that the electrostatistically dipolar nature of the catalytic domain is substantially modified upon activation, with cleavage of the negatively charged activation peptide leaving behind a largely hydrophobic face in factor IXa. While the backbone atoms of the catalytic residues have little relative movement, nearby loops are found that do move. The presence or absence of these changes likely defines specificity.

p53 mutants exhibiting enhanced transcriptional activation and altered promoter selectivity are revealed using a sensitive, yeast-based functional assay.

Changes in promoter specificity and binding affinity that may be associated with p53 mutations or post-translational modifications are useful in understanding p53 structure/function relationships and categorizing tumor mutations. We have exploited variable expression of human p53 in yeast to identify mutants with novel phenotypes that would correspond to altered promoter selectivity and affinity. The p53 cDNA regions coding for the DNA binding and tetramerization domains were subjected to random PCR mutagenesis and were cloned directly by recombination in yeast into a vector with a GAL1 promoter whose level of expression could be easily varied. p53 variants exhibiting higher than wild type levels of transactivation (supertrans) for the RGC responsive element were identified at low level of p53 protein expression. All the p53 mutants obtained with this screen were located in the DNA binding domain. Two out of 17 supertrans mutants have been found in tumors. Six mutations were in the L1 loop region between amino acids 115 and 124. The transactivation potential of a panel of supertrans p53 mutants on different promoters was evaluated using the p53 responsive elements, RGC, PIG3, p21 and bax. Although all mutants retained some activity with all promoters, we found different patterns of induction based on strength and promoter specificity. In particular none of the mutants was supertrans for the p21 responsive element. Interestingly, further analysis in yeast showed that the transactivation function could be retained even in the presence of dominant-negative p53 tumor mutations that could inhibit wild type p53. Five mutants were also characterized in human cells in terms of growth suppression and transactivation of various promoters. These novel supertrans p53 mutants may be useful in studies aimed at dissecting p53 downstream pathways, understanding specific interactions between p53 and the DNA, and could replace wild type p53 in cancer gene therapy protocols. The approach may also prove useful in identifying p53 tumor mutations.

Biased distribution of inverted and direct Alus in the human genome: implications for insertion, exclusion, and genome stability.

Alu sequences, the most abundant class of large dispersed DNA repeats in human chromosomes, contribute to human genome dynamics. Recently we reported that long inverted repeats, including human Alus, can be strong initiators of genetic change in yeast. We proposed that the potential for interactions between adjacent, closely related Alus would influence their stability and this would be reflected in their distribution. We have undertaken an extensive computational analysis of all Alus (the database is at http://dir.niehs.nih.gov/ALU) to better understand their distribution and circumstances under which Alu sequences might affect genome stability. Alus separated by <650 bp were categorized according to orientation, length of regions sharing high sequence identity, distance between highly identical regions, and extent of sequence identity. Nearly 50% of all Alu pairs have long alignable regions (>275 bp), corresponding to nearly full-length Alus, regardless of orientation. There are dramatic differences in the distributions and character of Alu pairs with closely spaced, nearly identical regions. For Alu pairs that are directly repetitive, approximately 30% have highly identical regions separated by <20 bp, but only when the alignments correspond to near full-size or half-size Alus. The opposite is found for the distribution of inverted repeats: Alu pairs with aligned regions separated by <20 bp are rare. Furthermore, closely spaced direct and inverted Alus differ in their truncation patterns, suggesting differences in the mechanisms of insertion. At larger distances, the direct and inverted Alu pairs have similar distributions. We propose that sequence identity, orientation, and distance are important factors determining insertion of adjacent Alus, the frequency and spectrum of Alu-associated changes in the genome, and the contribution of Alu pairs to genome instability. Based on results in model systems and the present analysis, closely spaced inverted Alu pairs with long regions of alignment are likely at-risk motifs (ARMs) for genome instability.

Heparan sulfate biosynthesis: a theoretical study of the initial sulfation step by N-deacetylase/N-sulfotransferase.

Heparan sulfate N-deacetylase/N-sulfotransferase (NDST) catalyzes the deacetylation and sulfation of N-acetyl-D-glucosamine residues of heparan sulfate, a key step in its biosynthesis. Recent crystallographic and mutational studies have identified several potentially catalytic residues of the sulfotransferase domain of this enzyme (, J. Biol. Chem. 274:10673-10676). We have used the x-ray crystal structure of heparan sulfate N-sulfotransferase with 3'-phosphoadenosine 5'-phosphate to build a solution model with cofactor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and a model heparan sulfate ligand bound, and subsequently performed a 2-ns dynamics solution simulation. The simulation results confirm the importance of residues Glu(642), Lys(614), and Lys(833), with the possible involvement of Thr(617) and Thr(618), in binding PAPS. Additionally, Lys(676) is found in close proximity to the reaction site in our solvated structure. This study illustrates for the first time the possible involvement of water in the catalysis. Three water molecules were found in the binding site, where they are coordinated to PAPS, heparan sulfate, and the catalytic residues.

Modeling zymogen protein C.

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I.

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.

Molecular dynamics simulations of the d(CCAACGTTGG)(2) decamer: influence of the crystal environment.

Molecular dynamics (MD) simulations of the DNA duplex d(CCAACGTTGG)(2) were used to study the relationship between DNA sequence and structure. Two crystal simulations were carried out; one consisted of one unit cell containing two duplexes, and the other of two unit cells containing four duplexes. Two solution simulations were also carried out, one starting from canonical B-DNA and the other starting from the crystal structure. For many helicoidal parameters, the results from the crystal and solution simulations were essentially identical. However, for other parameters, in particular, alpha, gamma, delta, (epsilon - zeta), phase, and helical twist, differences between crystal and solution simulations were apparent. Notably, during crystal simulations, values of helical twist remained comparable to those in the crystal structure, to include the sequence-dependent differences among base steps, in which values ranged from 20 degrees to 50 degrees per base step. However, in the solution simulations, not only did the average values of helical twist decrease to approximately 30 degrees per base step, but every base step was approximately 30 degrees, suggesting that the sequence-dependent information may be lost. This study reveals that MD simulations of the crystal environment complement solution simulations in validating the applicability of MD to the analysis of DNA structure.

Abasic sites induce triplet-repeat expansion during DNA replication in vitro.

The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.

Molecular dynamics simulations of biomolecules: long-range electrostatic effects.

Current computer simulations of biomolecules typically make use of classical molecular dynamics methods, as a very large number (tens to hundreds of thousands) of atoms are involved over timescales of many nanoseconds. The methodology for treating short-range bonded and van der Waals interactions has matured. However, long-range electrostatic interactions still represent a bottleneck in simulations. In this article, we introduce the basic issues for an accurate representation of the relevant electrostatic interactions. In spite of the huge computational time demanded by most biomolecular systems, it is no longer necessary to resort to uncontrolled approximations such as the use of cutoffs. In particular, we discuss the Ewald summation methods, the fast particle mesh methods, and the fast multipole methods. We also review recent efforts to understand the role of boundary conditions in systems with long-range interactions, and conclude with a short perspective on future trends.

Residues in the alphaH and alphaI helices of the HIV-1 reverse transcriptase thumb subdomain required for the specificity of RNase H-catalyzed removal of the polypurine tract primer.

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the alphaH and alphaI helices in the thumb subdomain play in specific RNase H cleavage at the 3'-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3'-end. Further analysis of alphaH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.