Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA.
We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.
Bonner
C A
CA
Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340.
Randall
S K
SK
Rayssiguier
C
C
Radman
M
M
Eritja
R
R
Kaplan
B E
BE
McEntee
K
K
Goodman
M F
MF
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004257:DNA Polymerase II / Q000302:isolation & purification*; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009268:Nalidixic Acid / Q000494:pharmacology; D009690:Nucleic Acid Conformation
D009268:Nalidixic Acid; D004247:DNA; D004257:DNA Polymerase II