Characterization of amino acid substitutions that severely alter the DNA repair functions of Escherichia coli endonuclease IV.
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.
Yang
X
X
ICRF, Clare Hall Laboratories, South Mimms, Hertsfordshire EN6 3LD, United Kingdom.
Tellier
P
P
Masson
J Y
JY
Vu
T
T
Ramotar
D
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004352:Drug Resistance, Microbial; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; C057799:3'-phosphoglycoaldehyde diesterase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase