Characterization of Amino Acid Substitutions That Severely Alter the DNA Repair Functions of Escherichia coli Endonuclease IV

Abstract

Escherichia coli endo IV is a bifunctional DNA repair protein, i.e., possessing both apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase activities. The former activity cleaves AP sites, whereas the latter one removes a variety of 3'-blocking groups present at single-strand breaks in damaged DNA. However, the precise reaction mechanism by which endo IV cleaves DNA lesions is unknown. To probe this mechanism, we have identified eight amino acid substitutions that alter endo IV function in vivo. Seven of these mutant proteins are variably expressed in E. coli and, when purified, show a 10-60-fold reduction in both AP endonuclease and 3'-diesterase activities. The most severe defect was observed with the one remaining mutant (E145G) that showed normal protein expression. This mutant has lost the ability to bind double-stranded DNA and showed a dramatic 150-fold reduction in enzymatic activities. We conclude that the AP endonuclease and the 3'-diesterase activities of endo IV are associated with a single active site, that is perhaps remote from the DNA binding domain.

Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

“…The crystal structure of Nfo complexed to duplex DNA containing an AP site shows that three Zn 2ϩ ions are bound in the protein by conserved residues that cluster at the center of a crescent-shaped cavity (6). Site-directed mutagenesis of the con- Nfo-H69A Nfo-G149D served amino acid residues support participation of Zn 2ϩ ions in catalysis of phosphodiester bond cleavage (18). However, based on analysis of the Nfo⅐DNA structure and present data, we speculated that Zn1 atom, coordinated by the side chains of H69, H109, and E145, might be dispensable for cleavage of an AP site.…”

Saccharomyces cerevisiae Apn1 mutation affecting stable protein expression mimics catalytic activity impairment: Implications for assessing DNA repair capacity in humans

“…This mutant was of particular interest because V156 is a conserved residue (Fig. 1, indicated by asterisk) for which a mutation at the corresponding residue in the E.coli endo IV mutant V143E has been previously shown to exhibit catalytic deficiency and decreased functional repair capacity that is independent of cellular protein levels [54]. This provided the opportunities to elucidate structure-function relationships for endonuclease IV family endonucleases and to explore how this deficiency manifests itself in eukaryotic cells.…”

“…Furthermore, they share 100% amino acid identity with respect to the nine conserved metal binding amino acid residues that comprise the endo IV active site (Fig. 1A, bolded, underlined text), which together are important for DNA incision activity [20, 23, 54]. …”

Pir1p Mediates Translocation of the Yeast Apn1p Endonuclease into the Mitochondria To Maintain Genomic Stability

“…In a separate experiment, the level of AP endonuclease activity was measured in the nuclear extracts using a synthetic substrate containing AP sites (48). After 2 h of induction, AP endonuclease levels in the extracts derived from purified nuclei isolated from the four strains SEY6210/pGFP-APN1, RVY1/ pGFP-APN1, SEY6210/pGFP-IMP2, and RVY1/pGFP-IMP2 were 169.1, 1,015.6, 42.3, and 189.2 U per mg of protein, respectively.…”

“…A typical assay consists of 1 pmol of substrate in 25 l of reaction mix containing 50 mM HEPES-KOH (pH 7.6), 50 mM KCl, 1 mM EDTA, 100 g of bovine serum albumin, and the indicated concentration of protein extracts or purified protein. One unit of enzyme cleaves 1 pmol of the substrate/min under the standard reaction condition (48).…”