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The tyrosine‐6 hydroxyl of γδ resolvase is not required for the DNA cleavage and rejoining reactions
Author(s) -
Leschziner Andres E.,
Boocock Martin R.,
Grindley Nigel D. F.
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.tb02356.x
Subject(s) - tn3 transposon , site specific recombination , dna , tyrosine , cleavage (geology) , biology , serine , cleave , recombinase , mutant , residue (chemistry) , nucleophile , stereochemistry , biochemistry , holliday junction , recombination , chemistry , dna repair , enzyme , transposable element , catalysis , gene , paleontology , fracture (geology)
Summary Site‐specific recombinases of the resolvase and DNA invertase family all contain a tyrosine residue close to the N ‐terminus, and four residues away from a serine that has been implicated in catalysis of DNA strand breakage and reunion. To examine the role of this tyrosine in recombination, we have constructed a mutant of γδ resolvase in which the tyrosine (residue 6) is replaced by phenytalanine. Characterization of the Y6F mutant protein in vitro indicated that although it was highly defective in recombination, it could cleave ONA at the cross‐over site, form a covalent resolvase‐DNA complex and rejoin the cleaved cross‐over site (usually restoring the parental site). These data rule out a direct role of the Tyr‐6 hydroxyl as the nucleophile In the DNA cleavage reaction and strengthen the conclusion that this nucleophile is the nearby invariant serine residue, Ser‐10. We conclude that Tyr‐6 is essential for fully coordinated strand cleavage and exchange, but is dispensable for individual strand cleavage and religation reactions.