Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase

Freeman-Wittig, M. J.; Vinocour, Michelle; Lewis, Roger

doi:10.1021/bi00358a047

Cited by 10 publications

(3 citation statements)

References 22 publications

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“…As far as is known, the latter do not polymerase-endonuclease" arrangement (28,29 5). The amino-terminal residue of RSV shown in the alignment is known to be the amino-terminal residue of that endonuclease (7), as is depicted in Fig.…”

Section: Discussionmentioning

confidence: 98%

Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes.

Johnson¹,

McClure²,

Feng³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

469

259

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A computer analysis of the amino acid sequences from the putative gene products of retroviral pol genes has revealed a 150-residue segment that is homologous with the ribonuclease H of Escherichia coli. The segment occurs at the carboxyl terminus of the region assigned to the 90-kDa reverse transcriptase polypeptide. In contrast, a section nearer the amino terminus of this sequence can be aligned with nonretroviral polymerases. (5), and an endonuclease ("integrase") that is essential for the integration of the newly synthesized DNA into the host genome (6).The pol gene of retroviruses is expressed initially as a gag-pol precursor that is proteolytically processed to a number of small gag proteins, an approximately 90-kDa protein encompassing both RNA-directed DNA polymerase (reverse transcriptase) and ribonuclease H activities, and, finally, a 40-kDa fragment with endonuclease activity (7). Several reports have presented evidence that the ribonuclease H activity of the 90-kDa reverse transcriptase portion is associated with the amino-terminal end of that protein, and by implication, that the DNA polymerase activity is at the carboxyl-terminal end. These conclusions are based on experiments involving deletion mutants (2), on the one hand, and antibodies to synthetic peptides modeled on the putative sequences, on the other (3).We now suggest that the opposite must be true: the ribonuclease H activity should be situated at the carboxyl terminus, and the DNA polymerase, at the amino terminus. We draw this conclusion on the basis of comparisons of the retroviral sequences with those of nonviral enzymes of similar function. In this regard, we have uncovered a significant resemblance between a 150-residue segment at the carboxyl-terminal end of the 90-kDa fragment and the reported sequence of a ribonuclease H from Escherichia coli. We also provide an alignment of a segment near the amino terminus of the 90-kDa polypeptide with highly conserved sequences from many other polymerases, including the a subunit of E. coli DNA-directed RNA polymerase. Finally, there is a distinctive sequence in the endonuclease sequence that is characteristic of a zinc-binding segment. METHODSThe sequences used were taken from the 1985 version of NEWAT (8) identical (Fig. 1). Binary comparison of each of the retroviral sequences with the E. coliribonuclease H sequence, followed by statistical evaluation by a randomization method, gave authentic alignment scores from 4 to 10 standard deviations above the means of the jumbled comparisons. The cumulative weight of the multiple alignment (Fig. 2) further bears out the significance of the overall relationship. That the polymerase portion of the viral reverse transcriptase system must encompass the amino-terminal portion of the 90-kDa fragment is established by the alignment shown in Fig. 3. The key region here involves a sector previously shown by Kamer and Argos (20) to be present in a number of nonretroviral polymerases; these consistently have two aspartic acid residues surrounded by...

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Section: Discussionmentioning

confidence: 98%

Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes.

Johnson¹,

McClure²,

Feng³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

469

259

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“…injection, which increases bioavailability of folpet and limits the neutralizing actions of cellular thiols� Because i.p. injection bypasses the normal routes of exposure, it does not simulate anticipated human exposure� DNA polymerases, similar to histones, are integrally linked to DNA integrity� In vitro biochemical investigations have demonstrated that captan inhibits DNA polymerase by competing for the same site as the DNA while not interfering with the fidelity of the DNA polymerase I copy of the DNA template (Dillwith and Lewis, 1982)� Using captan as an inhibitor of viral reverse transcriptase, the differential effects of captan on DNA polymerase and RNase H activity could be determined Simmon et al, 1977Reassessment Jones et al 1984 In vitro Simmon et al, 1977Reassessment Jones et al 1984 In vitro (Freeman-Wittig et al�, 1986)� RNase H activity was 10-fold more sensitive to captan than either DNA-dependent or RNAdependent DNA polymerase� Based on the observation that dithiothreitol prevented captan inhibition, it was concluded that the trichloromethylthio moiety of captan was involved in the inhibitory action (Freeman-Wittig et al�, 1986)� Captan was subsequently found to be active at but not limited to the nucleoside triphosphate binding site and DNA binding site of the RNA polymerase (Luo and Lewis, 1992)� A number of in vitro studies have been conducted with the goal of elucidating key elements associated with folpet and captan's mutagenicity� The reactivity of folpet, captan, and thiophosgene is such that within an in vitro environment reactions will occur with available thiols� The in vitro environment is distinct from the intact animal� Although these mechanistic studies speak to possible modes of mutagenic action, they are not performed in systems comparable to the in vivo setting where captan and folpet mutagenicity is absent�…”

Section: Dna-specific Proteinsmentioning

confidence: 99%

Genetic toxicology of folpet and captan

Arce¹,

Gordon²,

Cohen

et al. 2010

Critical Reviews in Toxicology

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Folpet and captan are fungicides whose genotoxicity depends on their chemical reaction with thiols. Multiple mutagenicity tests have been conducted on these compounds due to their positive activity in vitro and their association with gastrointestinal tumors in mice. A review of the collective data shows that these compounds have in vitro mutagenic activity but are not genotoxic in vivo. This dichotomy is primarily due to the rapid degradation of folpet and captan in the presence of thiol-rich matrices typically found in vivo. Genotoxicity has not been found in the duodenum, the mouse tumor target tissue. It is concluded that folpet like captan presents an unlikely risk of genotoxic effects in humans.

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“…Nevertheless, there have been hints that the two activities may be separable. Limited proteolysis has permitted the recovery of fragments exhibiting RNase H activity in the absence of DNA polymerase (19), and some chemicals exhibit differential inhibitory activity (20)(21)(22)(23).Recently, a computer-aided comparison of the amino acid sequences of various reverse transcriptases and those of other enzymes has led to a proposal for the functional organization of the viral proteins (24). The positions of the similarities suggested that the N-terminal part of reverse transcriptase should contain the DNA polymerase activity, whereas the C-terminal part should contain RNase H. To map the two activities and to determine whether the expression of each activity in the absence of its partner could be achieved, we have carried out a mutational analysis of, a cloned copy of a viral reverse transcriptase gene (25).…”

mentioning

confidence: 99%

Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities.

Tanese

Goff

1988

Proc. Natl. Acad. Sci. U.S.A.

197

129

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The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNADNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity.Soon after a retrovirus enters a permissive cell, the' virion core directs the synthesis of a double-stranded DNA copy of the encapsidated genomic RNA. The reaction is catalyzed by the virus-encoded enzyme reverse transcriptase, which is carried into the cell within the virion particle (1,2). Although the overall process of reverse transcriptase is complex, the general outline of the reaction has been deduced by the study of in vitro systems (3). Synthesis of the first DNA strand is initiated with a tRNA primer and proceeds by copying RNA to ultimately form full-length'viral DNAs. In the course of first-strand DNA synthesis, the genomic RNA is degraded (4); that degradation exposes the first DNA strand for use as template and also forms the primer RNA for initiation of the second DNA strand (5). The final product is a duplex DNA free of RNA, after the primer RNAs at the 5' ends of each DNA strand are removed (6, 7). Reverse transcriptase enzymes purified from virion cores exhibit many of the activities thought to be needed for these reactions (8). In particular, all known 'reverse transcriptases exhibit two catalytic activities: a DNA polymerase activity and an associated RNase activity (9-11). The DNA polymerase activity can extend the 3' end of a primer.and copy either RNA or DNA templates, as is required to form the first and second strands of the viral DNA. The RNase activity, termed RNase H, degrades RNA only when it is in the form of an RNA-DNA hybrid duplex; single-stranded RNA and RNARNA duplexes are resistant to degradation (9,10,12). This activity can account for the degradation of the viral RNA, for secondstrand primer formation, and for primer removal.All reverse transcriptases are formed by proteolytic processing from a large polyprotein precursor (13), but the mature enzymes from different virus families show different subunit structures. The enzyme of avian viruses is an a-,8 heterodimer; the larger f8 subunit contains all of the sequences in the a subunit but includes extra sequences at the C terminus (14). Chromatographic separation of the subunits has shown that each subunit has both DNA polymerase and RNase H activities (15). The enzyme from murine and feline viruses is a simple monomer that ...

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Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase

Cited by 10 publications

References 22 publications

Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes.

Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes.

Genetic toxicology of folpet and captan

Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities.

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