Sharon P. Moore

A Ty1 Integrase Nuclear Localization Signal Required for Retrotransposition

ABSTRACT Ty1 retrotransposition in Saccharomyces cerevisiaerequires integrase (IN)-mediated insertion of Ty1 cDNA into the host genome. The transposition components are assembled in the cytoplasm and must cross the nuclear envelope to reach the genomic target, since, unlike animal cell nuclear membranes, the yeast cell nuclear membrane remains intact throughout the cell cycle. We have identified a bipartite nuclear localization signal (NLS) in IN required for Ty1 transposition (Ty1 IN) that directs IN to the nucleus. Mutations in the NLS that specifically abolish nuclear localization inactivate transpositional integration but do not affect reverse transcription, protein processing, or catalytic activity in vitro. No additional Ty1-encoded proteins are required for IN nuclear localization. Intragenic complementation experiments suggest that Ty1 IN functions as a multimer and contains two distinct domains, one required for integration and the other for nuclear localization. Nuclear targeting of the preintegration complex by an IN NLS may prove to be a general strategy used by retrotransposons and retroviruses that infect nondividing cells.

The Genomic RNA in Ty1 Virus-Like Particles Is Dimeric

ABSTRACT The yeast retrotransposon Ty1 resembles retroviruses in a number of important respects but also shows several fundamental differences from them. We now report that, as in retroviruses, the genomic RNA in Ty1 virus-like particles is dimeric. The Ty1 dimers also resemble retroviral dimers in that they are stabilized during the proteolytic maturation of the particle. The stabilization of the dimer suggests that one of the cleavage products of TyA1 possesses nucleic acid chaperone activity.

S-Phase Checkpoint Pathways Stimulate the Mobility of the Retrovirus-Like Transposon Ty1

ABSTRACT The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101Δ mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101Δ mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

Analysis of a Ty1-less variant of Saccharomyces paradoxus: the gain and loss of Ty1 elements

Because Ty elements transpose through an RNA intermediate, element accumulation through retrotransposition must be regulated or offset by element loss to avoid uncontrolled genome expansion. Here we examine the fate of Ty sequences in Saccharomyces strain 337, a strain that is reported to lack Ty1 and Ty2 elements, but contains remnant solo long terminal repeats (LTRs). Although strain 337 was initially classified as Saccharomyces cerevisiae, our work indicates that this strain is more closely related to S. paradoxus. Several degenerate Ty1 and Ty2 LTRs were mapped to the same insertion sites as full‐length Ty1 and Ty2 elements in S. cerevisiae, suggesting that this strain lost Ty elements by LTR–LTR recombination. Southern analysis indicates that strain 337 also lacks Ty4 and Ty5 elements. We estimated the rates of element gain and loss in this strain by introducing a single transposition‐competent Ty1 element. The results indicate that Ty1 retrotransposition occurs at a much higher rate than elimination, suggesting that copy‐number‐dependent co‐factors or environmental conditions contribute to the loss of Ty elements in this genome. Copyright

Retrotransposon Suicide: Formation of Ty1 Circles and Autointegration via a Central DNA Flap

ABSTRACT Despite their evolutionary distance, the Saccharomyces cerevisiae retrotransposon Ty1 and retroviruses use similar strategies for replication, integration, and interactions with their hosts. Here we examine the formation of circular Ty1 DNA, which is comparable to the dead-end circular products that arise during retroviral infection. Appreciable levels of circular Ty1 DNA are present with one-long terminal repeat (LTR) circles and deleted circles comprising major classes, while two-LTR circles are enriched when integration is defective. One-LTR circles persist when homologous recombination pathways are blocked by mutation, suggesting that they result from reverse transcription. Ty1 autointegration events readily occur, and many are coincident with and dependent upon DNA flap structures that result from DNA synthesis initiated at the central polypurine tract. These results suggest that Ty1-specific mechanisms minimize copy number and raise the possibility that special DNA structures are a targeting determinant.

Correct Integration of Model Substrates by Ty1 Integrase

ABSTRACT The retrovirus-like mobile genetic element of Saccharomyces cerevisiae, Ty1, transposes to new genomic locations via the element-encoded integrase (IN). Here we report that purified recombinant IN catalyzed correct integration of a linear DNA into a supercoiled target plasmid. Ty1 virus-like particles (VLPs) integrated donor DNA more efficiently than IN. VLP and IN-mediated insertions occurred at random sites in the target. Mg2+ was preferred over Mn2+ for correct integration, and neither cation enhanced nonspecific nuclease activity of IN. Products consistent with correct integration events were also obtained by Southern analysis. Recombinant IN and VLPs utilized many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.

Functional Analysis of N-Terminal Residues of Ty1 Integrase

ABSTRACT The Ty1 retrotransposon of Saccharomyces cerevisiae is comprised of structural and enzymatic proteins that are functionally similar to those of retroviruses. Despite overall sequence divergence, certain motifs are highly conserved. We have examined the Ty1 integrase (IN) zinc binding domain by mutating the definitive histidine and cysteine residues and thirteen residues in the intervening (X32) sequence between IN-H22 and IN-C55. Mutation of the zinc-coordinating histidine or cysteine residues reduced transposition by more than 4,000-fold and led to IN and reverse transcriptase (RT) instability as well as inefficient proteolytic processing. Alanine substitution of the hydrophobic residues I28, L32, I37 and V45 in the X32 region reduced transposition 85- to 688-fold. Three of these residues, L32, I37, and V45, are highly conserved among retroviruses, although their effects on integration or viral infectivity have not been characterized. In contrast to the HHCC mutants, all the X32 mutants exhibited stable IN and RT, and protein processing and cDNA production were unaffected. However, glutathione S-transferase pulldowns and intragenic complementation analysis of selected transposition-defective X32 mutants revealed decreased IN-IN interactions. Furthermore, virus-like particles with in-L32A and in-V45A mutations did not exhibit substantial levels of concerted integration products in vitro. Our results suggest that the histidine/cysteine residues are important for steps in transposition prior to integration, while the hydrophobic residues function in IN multimerization.