Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein

Journal Article · Tue Apr 23 04:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00230a004· OSTI ID:5396352

Myles, G M; Sancar, A ^[1]; Hearst, J E ^[2]

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1. Univ. of North Carolina School of Medicine, Chapel Hill (USA)
2. Univ. of California, Berkeley (USA)

UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. The authors have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646 mutant, and 95% in the K37A-K646A double mutant. They find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA){sub 2}(UvrB){sub 1} complexes. They conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.

• OSTI ID: 5396352
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:16; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

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AMINO ACID SEQUENCE
ATP-ASE
BACTERIA
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