Functional analysis of N-terminal residues of ty1 integrase.
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.
Moore
Sharon P
SP
Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA. moores@ncifcrf.gov
Garfinkel
David J
DJ
D020816:Amino Acid Motifs; D001665:Binding Sites; D017124:Conserved Sequence; D003545:Cysteine; D006639:Histidine; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D055503:Protein Multimerization; D018626:Retroelements; D012190:Retroviridae; D012441:Saccharomyces cerevisiae; D016662:Virus Integration; D015032:Zinc / Q000378:metabolism
D018626:Retroelements; D006639:Histidine; D019426:Integrases; D015032:Zinc; D003545:Cysteine