The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro.

TitleThe Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro.
Publication TypeJournal Article
Year of Publication2000
AuthorsMamun, AA al, Yadava, RS, Ren, L, Humayun, MZ
JournalMol Microbiol
Volume38
Issue2
Pagination368-80
Date Published2000 Oct
ISSN0950-382X
KeywordsBacterial Proteins, Bacteriophage M13, Cytosine, DNA Polymerase beta, DNA Polymerase I, DNA Polymerase II, DNA Replication, DNA, Single-Stranded, DNA, Viral, DNA-Directed DNA Polymerase, Escherichia coli, Escherichia coli Proteins, Mutagenesis, Site-Directed, Mutagens, SOS Response (Genetics), Ultraviolet Rays
Abstract

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) 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 epsilonC 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.

Alternate JournalMol. Microbiol.
PubMed ID11069662
Grant ListCA73026 / CA / NCI NIH HHS / United States
GM58153 / GM / NIGMS NIH HHS / United States