Abstract
DNA lesions that block replication can be bypassed in Escherichia coli by a special DNA synthesis process termed translesion replication. This process is mutagenic due to the miscoding nature of the DNA lesions. We report that the repair enzyme formamido-pyrimidine DNA glycosylase and the general DNA damage recognition protein UvrA each inhibit specifically translesion replication through an abasic site analog by purified DNA polymerases I and II, and DNA polymerase III (alpha subunit) from E. coli. In vivo experiments suggest that a similar inhibitory mechanism prevents at least 70% of the mutations caused by ultraviolet light DNA lesions in E. coli. These results suggest that DNA damage-binding proteins regulate mutagenesis by a novel mechanism that involves direct inhibition of translesion replication. This mechanism provides anti-mutagenic defense against DNA lesions that have escaped DNA repair.
Highlights
Mutations caused by DNA-damaging agents play a key role in cancer via activation of oncogenes and inactivation of tumor suppressor genes [1]
Two excision repair mechanisms are known for DNA damage: nucleotide excision repair (NER),1 which operates on a wide variety of DNA lesions [3], and base excision repair (BER) which is more limited in its range of substrate specificity [4]
In the bacterium Escherichia coli, the protein UvrA is responsible for the recognition of a broad range of DNA lesions that are repaired by NER, whereas in BER a series of DNA glycosylases with a much narrower range of specificity recognize and excise damaged or foreign bases from DNA [2]
Summary
Materials—UvrA and RecA were purified as described [11,12,13]. DNA polymerase I (6000 units/mg) was obtained from Boehringer Mannheim. DNA polymerase II, purified according to Ref. 14, was a gift from M. Goodman (University of Southern California, Los Angeles, CA), and DNA polymerase III (␣ subunit), purified as described [15], was a gift from M. All template molecules contain the abasic site analog (Ͼ99.98%), as deduced from the complete arrest of DNA synthesis at the lesion when bypass was assayed with pol I in the presence of 0.1 M KCl (Ͻ0.02% bypass, representing the limit of detection; Ref. 18). The in vitro replication reaction mixture (25 or 50 l) contained 20 mM Tris1⁄7HCl, pH 7.5, 8 g/ml bovine serum albumin, 5 mM dithiothreitol, 0.1 mM EDTA, 4% glycerol, 10 mM MgCl2, 1 mM ATP and. WBY130 was prepared by moving the ⌬uvrA::cat mutation from strain MH1 (obtained from P. van de Putte, Leiden University, Leiden, The Netherlands) to AB1157. Hisϩ mutants were assayed as described above for UV mutagenesis
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