Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding

Journal Article · Tue Mar 22 04:00:00 EST 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00406a007· OSTI ID:6908277

Stump, D G; Lloyd, R S

T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.

• Research Organization: Vanderbilt Univ. School of Medicine, Nashville, TN (USA)
• OSTI ID: 6908277
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:6; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

560130* -- Radiation Effects on Microorganisms
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACID SEQUENCE
AMINO ACIDS
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CARBOXYLIC ACIDS
DNA
DNA REPAIR
DNA-ASE
ELECTROMAGNETIC RADIATION
ENDONUCLEASES
ENZYMES
ESCHERICHIA COLI
ESTERASES
FUNCTIONS
GENES
HYDROLASES
HYDROXY ACIDS
KINETICS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PYRIMIDINE DIMERS
RADIATIONS
REACTION KINETICS
RECOVERY
REPAIR
SURVIVAL CURVES
TYROSINE
ULTRAVIOLET RADIATION