The evolution of the long and short repetitive DNA sequences in sea urchins.

The rates of evolution of purified long and short repetitive DNA sequences were examined by hybridisation analysis between the DNAs from several species of sea urchins. We find that the rates of nucleotide substitution are very comparable within mutually retained sequences for the two classes of repetitive DNA. The loss of hybridisable sequences between species also occurs at similar rates among both the short and long repetitive DNA sequences. Between species that separated less than 50 million years ago, hybridisable short repetitive sequences are lost all through the spectrum of reiteration frequencies. The long repeats contain a few sequences which are highly conserved within all of the species examined, and which amount to approximately 1% of the total genome. The short repetitive class, on the other hand, does not seem to contain any such highly conserved elements. The long repetitive sequences internally appear to contain short 'units' of reiteration, which may comprise families within the long repetitive class. We find no evidence to indicate that the majority of long and short repetitive sequences evolve by different mechanisms or at different rates.

Characterization of long and short repetitive sequences in the sea urchin genome.

Long and short repetitive sequences were purified from the DNA of Paracentrotus lividus under conditions designed to optimize the yield of complete, end to end sequences. Double-stranded long repeat DNA prepared in this manner ranged in length from approximately 3000 to 15 000 nucleotide pairs with average sizes of approximately 6000 base pairs. In the electron microscope, long repeat DNA was observed to possess continuous sequences that often appeared to be terminated by one or more loops and/or fold backs. Long repeat DNA sequences, resheared to 300 base pairs, were found to have an average melting point identical to that for sheared native DNA. Thus, the reassociated duplexes of long repetitive DNA seem to possess very few mismatched base pairs. Reassociation kinetic analyses indicate that the majority of the long repeat sequences are reiterated only 4--7 times per haploid amount of DNA. Melt-reassociation analyses of short repetitive DNA, at several criteria, support the previously held concept that these sequences belong the sets or families of sequences which are inexact copies of one another. Our studies also support hypotheses suggesting that short repetitive sequences belong to families which may have arisen via distinct salttatory events. The relationships between long and short repetitive DNA sequences are considered with respect to widely held concepts of their sequence organization, evolution, and possible functions within eucaryotic genomes. A model for the possible organization of short repeats within long repetitive DNA sequences is also presented.

Substitution of lysine for arginine at position 199 of a hypoxanthine phosphoribosyltransferase interferes with binding of the primary substrate to the active site.

Lysine was substituted for a conserved arginine at position 199 of the schistosomal hypoxanthine phosphoribosyltransferase (HPRT). This resulted in a > or = 35-fold increase in the K(M) for binding phosphoribosyl-pyrophosphate (PRPP). The possible functional role of R199 in tertiary structure, as well as in the binding of PRPP, is interpreted in the context of the reported three dimensional structure for the human HPRT.

Limited proteolysis of a trypanosomal hypoxanthine phosphoribosyltransferase yields crystals that diffract X-rays to near atomic resolution.

Two crystal forms of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi were grown and characterized. Proteolytic modification at the C-terminus of the recombinant enzyme yielded monoclinic crystals that diffract X-rays to higher resolution than the original, trigonal crystal form. Data from the monoclinic crystal form enabled determination of the crystal structure for the trypanosomal HPRT to 1.4 A resolution.

Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase.

Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch-Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than pyrophosphate (PP(i)), for which there was a 5.8-fold increase. In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.

Sequence of human protein serine/threonine phosphatase 1 gamma and localization of the gene (PPP1CC) encoding it to chromosome bands 12q24.1-q24.2.

Complementary DNA encoding a catalytic subunit of protein phosphatase 1, termed PP1 gamma, was isolated from a human teratocarcinoma library. The sequence suggests that alternative splicing produces two forms of PP1 gamma, designated PP1 gamma 1 and PP1 gamma 2, which differ in their C-termini. The gene for human PP1 gamma (PPP1CC) was localized to chromosome 12 by analysis of somatic cell hybrid DNA and mapped to bands q24.1-q24.2 by in situ hybridisation. These data show that although PP1 gamma 1 and PP1 gamma 2 are 94% and 93% identical to PP1 alpha respectively, the PP1 gamma gene is not closely linked to the PP1 alpha gene, which has been mapped to chromosome 11.

Comparing the human and schistosomal hypoxanthine-guanine phosphoribosyltransferases by circular dichroism.

The hypoxanthine-guanine phosphoribosyltransferases (HGPRTases) of human and the parasitic trematode, Schistosoma mansoni, are of biomedical importance. The conformations of these two enzymes were studied by circular dichroism (CD). The schistosomal HGPRTase is estimated to contain 27% alpha-helix and 30% beta-structure. This result is consistent with what is predicted from a tertiary model (Craig, S.P., Cohen, F.E., Yuan, L., McKerrow, J.H. and Wang, C.C. (1991) in Molecular & Immunological Aspects of Parasitism (Wang, C.C., ed.), pp. 122-138, Am. Assoc. Adv. Sci., Washington DC, USA), which proposes that the enzyme is an alpha/beta barrel protein. The human enzyme is estimated to contain 21% alpha-helix and 53% beta-form. The two enzymes are different in their thermostability. The human enzyme remains active after being heated to 85 degrees C for 15 min, while the schistosomal enzyme only retains its activity at temperature below 65 degrees C. The transition temperature (T1/2) of the schistosomal HGPRTase was determined by CD measurement to be 57.5 degrees C. One of the enzyme substrates, phosphoribose pyrophosphate (PRPP), stabilizes the HGPRTases by preventing the human enzyme from unfolding at 85 degrees C and partially protecting the schistosomal enzyme from unfolding at 65 degrees C. It is suggested that the amino-acid substitutions in the human enzyme improve the spatial structure and stability of its alpha-helices, which may lead to an enhanced thermostability.

Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi.

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.

Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding.

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

Genome organization and ploidy number in Trypanosoma cruzi.

Trypanosoma cruzi total DNA was analyzed by DNA:DNA reassociation kinetics. The nonlinear least-squares computer solution could reasonably be fitted to three second-order kinetic components. The highly repetitive, middle repetitive, and single copy components comprised 9, 35 and 49% of the genome, respectively. The single copy sequences showed a kinetic complexity of approximately 4 times that of Escherichia coli and of some 11,000 average-sized structural genes. The repetitive sequences (about 6900) presented the long-period pattern of interspersion with a modal length of 7800 bases. The kinetic complexity of total DNA was compatible with a value of at least a diploid genome per cell.