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The Escherichia coli UVM response is accompanied by an SOS‐independent error‐prone DNA replication activity demonstrable in vitro
Author(s) -
Al Mamun Abu Amar M.,
Yadava Ramesh S.,
Ren Li,
Humayun M. Zafri
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.02136.x
Subject(s) - biology , microbiology and biotechnology , dna replication , dna polymerase , dna , dna polymerase ii , dna polymerase i , sos response , mutagenesis , dna polymerase delta , escherichia coli , mutation , dna damage , polymerase chain reaction , genetics , gene , reverse transcriptase
UVM is an SOS‐independent inducible response characterized by elevated mutagenesis at a site‐specific 3, N 4 ‐ethenocytosine (εC) residue borne on M13 single‐stranded DNA transfected into Escherichia coli cells pretreated with DNA‐damaging agents. By constructing and using E. coli strain AM124 ( polA polB umuDC dinB lexA1 [Ind–]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the ‘non‐replicative’ DNA polymerases, namely DNA polymerase I ( polA ), II ( polB ), IV ( dinB ) and V ( umuDC ). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA‐damaging agents. To address the question as to whether the UVM response is accompanied by an error‐prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single‐stranded DNA bearing a site‐specific lesion is converted to circular double‐stranded replicative‐form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo . The newly synthesized (minus) DNA strand is selectively amplified by ligation‐mediated polymerase chain reaction (LM‐PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site‐specific εC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM‐induced AM124 cells, with C → A mutations predominating over C → T mutations, a specificity similar to that observed in vivo . These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error‐prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.