Replication infidelity during a single cycle of Ty1 retrotransposition.

Abstract

Retroviruses undergo a high frequency of genetic alterations during the process of copying their RNA genomes. However, little is known about the replication fidelity of other elements that transpose via reverse transcription of an RNA intermediate. The complete sequence of 29 independently integrated copies of the yeast retrotransposon Tyl (173,043 nt) was determined, and the mutation rate during a single cycle of replication was calculated. The observed base substitution rate of 2.5 x 10-5 bp per replication cycle suggests that this intracellular element can mutate as rapidly as retroviruses. The pattern and distribution of errors in the Tyl genome is nonrandom and provides clues to potential in vivo molecular mechanisms of reverse transcriptase-mediated error generation, including heterogeneous RNase H cleavage of Tyl RNA, addition of terminal nontemplated bases, and transient dislocation and realignment of primer-templates.Overall, analysis of errors generated during Tyl replication underscores the utility of a genetically tractable model system for the study of reverse transcriptase fidelity.Reverse transcription is a notoriously error-prone process. Mutations occurring during retroviral replication include simple base substitutions and frameshifts as well as complex deletions, deletions with insertions, and hypermutations (1-6).This low fidelity of replication is presumed to be the basis for the rapid genome evolution of retroviruses as well as for the ability of human immunodeficiency virus to evade its host immune system and develop drug resistance. Biochemical studies using purified retroviral reverse transcriptases (RTs) have elucidated multiple potential error-generating mechanisms, including extension past mismatched bases (7-11), poor discrimination of dNTPs (12-14), lack of associated 3' to 5' exonuclease activities (9, 15), slippage of primer-templates (16,17), and addition of nontemplated bases at the ends of template strands (18,19).Retroviruses are the most highly visible members of a ubiquitous and varied confederation of genetic elements linked by their capacity to replicate by reverse transcription of an RNA intermediate (20,21). While recent biochemical analyses have revealed novel priming mechanisms associated with several nonretroviral , no study has addressed the rate or spectrum of errors occurring during a cycle of nonretroviral retrotransposition. Such studies are impeded by the polymorphic nature of multicopy elements in the genome (27, 28), their variable expression, and the difficulty of identifying events resulting from single rounds of replication. However, these problems could be obviated using a model system where transposition is limited to a single, marked element, where expression of the multiple endogenous copies is repressed, and where transposition events are trapped and prevented from secondary replication. The retrotransposon Tyl, from the yeast Saccharomyces cerevisiae, provides just such a system.Tyl is one of the most intensively studied retrotransposons. Its ...

Presence of wheat retrotransposons in Gramineae species and the origin of wheat retrotransposon families.

“…Taking into account the fact that the genomic clone carrying Tar1 was obtained by screening with high stringency washing and showed 96.7% homology to the ABD52 probe (see Results), the family 1 retrotransposons may show 96% or higher homology to the rice retrotransposons in the reverse transcriptase domain. The presumed high degree of homology suggests that the family 1 reverse transcriptase domain has long been under strong functional constraints in wheat and rice, because the mutation rates are extraordinarily high in retroelements including retrotransposons (Domingo and Holland, 1994;Gabriel et al, 1996).…”

Three Tnt1 Subfamilies Show Different Stress-Associated Patterns of Expression in Tobacco. Consequences for Retrotransposon Control and Evolution in Plants

“…Thus, we propose that the high sequence variability of Tnt1 and other plant retrotransposons, which is a consequence of the infidelity of the retrotransposition process (Gabriel et al, 1996) and the high copy number of these elements in plant genomes (Casacuberta et al, 1995), could have allowed their promoter sequences to evolve the optimum pattern of expression to be maintained in each host genome. This strategy obviously would be very different from that of Ty elements in yeast and could be a consequence of the important differences of the organization of these different host genomes.…”

The Tvv1 retrotransposon family is conserved between plant genomes separated by over 100 million years

“…In support of vertical radiation, pairwise comparisons of Tvv1 lTrs indicate that their pattern of similarity matches the successive divergence of the asterids and rosids followed by that of the clades within the rosids and the species within Solanum. nevertheless, the analysis may be biased by possible differing degrees of transpositional activity in the various species and clades, because for retrotransposons the error rates for replication via reverse transcription and as integral chromosomal components differ greatly (Gabriel et al 1996;Boutabout et al 2001;Abram et al 2010).…”

“…This life cycle is very prone to errors, estimated at 1.4 × 10 −5 mutations/bp/cycle, because both rnA polymerase II and reverse transcriptase lack proofreading activity (Gabriel et al 1996;Preston 1996;Abram et al 2010). Moreover, retrotransposon copies are replicated also as a part of the chromosome by DnA-dependent DnA polymerase; most copies are likely to accumulate mutations at the neutral rate.…”