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 (X 32 ) 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 X 32 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 X 32 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 X 32 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.The structural and functional similarities of the Ty retrotransposons of Saccharomyces to mammalian retroviruses suggest similar mechanisms for Ty transposition and retroviral integration. Like retroviruses, Ty elements are bound by long terminal repeats (LTR), contain two overlapping open reading frames analogous to retroviral gag and pol, and undergo proteolytic processing of a Gag/Pol fusion protein. The similarities between retrotransposons and retroviruses extend to the highly conserved sequence homologies in the catalytic active sites of the Pol proteins, protease (PR), RT,and IN (1,36,19,55,60). A noncatalytic sequence, the zinc binding domain (ZBD), is also conserved in the N-terminal regions of retroviral and retrotransposon INs (36). This domain, characterized by the sequence H-X 3-7 H-X 23-32 C-X 2 C, binds a single zinc ion (10, 37, 42, 67), which stabilizes the N-terminal region of IN and promotes formation of IN tetramers, thereby enhancing the enzyme's catalytic activity (37, 67).The IN catalytic domain has been well characterized in retroviruses (7, 34) and to a lesser extent in Ty1 (45, 54). The retroviral IN ZBD has also been studied extensively, initially by evaluating the effects of N-terminal deletions (53,8,11,12,16,23,59) or mutations of the histidine or cysteine residues (24, 57, 58) in assays which recapitulate biochemical activities of IN, i.e., 3Ј dinucleotide processing and integration of donor molecules representing v...

The Ty1 LTR-Retrotransposon of Budding Yeast,Saccharomyces cerevisiae

“…Ty1 IN does not repair the gaps, suggesting that either Ty1 RT or an unidentified cellular DNA repair protein performs this function. Ty1 IN has been shown to dimerize in vitro and by analogy with retroviral integrases, is thought to assemble as a tetramer on cDNA to form the pre-integration complex (PIC) [175]. …”

“…Alanine substitution of each of the four amino acids (H 17 H 22 C 55 C 58 ) of Ty1 IN ZBD motif results in IN instability and inefficient proteolytic processing, leading to substantially decreased cDNA levels and retrotransposition. Alanine substitution of additional hydrophobic residues between H 22 and C 55 , which are conserved between LTR-retrotransposons and retroviruses, alters IN-IN interaction, in vitro integration and in vivo retrotransposition [175]. …”

Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro

“…IN binds to the cDNA to form the pre-integration complex, which migrates into the nucleus where the cDNA is integrated into the host genome (6), preferentially upstream of Pol III-transcribed genes (7)(8)(9)(10) through a direct interaction between IN and Pol III (11,12). Previous studies suggest that, like retroviruses, Ty1 integration process is carried out by complexes composed of multiple copies of IN assembled on the LTR DNA ends, commonly called intasomes (13,14).…”

BUD22 Affects Ty1 Retrotransposition and Ribosome Biogenesis in Saccharomyces cerevisiae

“…Ty1-VLP purification: VLPs were prepared by differential centrifugation and sucrose gradient sedimentation as previously described (Eichinger and Boeke 1988;Moore and Garfinkel 2009). …”