Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3′–5′ exonuclease proofreading function

AUTOR(ES)

Vandewiele, Dominique

FONTE

The National Academy of Sciences

RESUMO

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD′2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3′–5′ exonuclease proofreading activity of the pol III ɛ-subunit also is disabled. TR was measured in isogenic uvrA6 ΔumuDC strains carrying the dominant negative dnaQ allele, mutD5, or ΔdnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T–T dimer. As expected, little TR was observed in the ΔumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated ΔumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind−) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for ≈70% of the TR, and another LexA-independent, responsible for the remaining ≈30%. Curiously, the ΔumuDC ΔdnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in ΔdnaQ strains, since introduction of spq-2 into the ΔumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041–6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the ΔumuDC mutD5 strain is unchanged by introduction of a ΔpolB mutation.

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