Functions of multiple exonucleases are essential for cell viability, DNA repair and homologous recombination in recD mutants of Escherichia coli

Abstract

Heterotrimeric RecBCD enzyme unwinds and resects a DNA duplex containing blunt double-stranded ends and directs loading of the strand-exchange protein RecA onto the unwound 3'-ending strand, thereby initiating the majority of recombination in wild-type Escherichia coli. When the enzyme lacks its RecD subunit, the resulting RecBC enzyme, active in recD mutants, is recombination proficient although it has only helicase and RecA loading activity and is not a nuclease. However, E. coli encodes for several other exonucleases that digest double-stranded and single-stranded DNA and thus might act in consort with the RecBC enzyme to efficiently promote recombination reactions. To test this hypothesis, I inactivated multiple exonucleases (i.e., exonuclease I, exonuclease X, exonuclease VII, RecJ, and SbcCD) in recD derivatives of the wild-type and nuclease-deficient recB1067 strain and assessed the ability of the resultant mutants to maintain cell viability and to promote DNA repair and homologous recombination. A complex pattern of overlapping and sometimes competing activities of multiple exonucleases in recD mutants was thus revealed. These exonucleases were shown to be essential for cell viability, DNA repair (of UV- and gamma-induced lesions), and homologous recombination (during Hfr conjugation and P1 transduction), which are dependent on the RecBC enzyme. A model for donor DNA processing in recD transconjugants and transductants was proposed.

Figure 1.

UV survival of the recB1067 recD mutant strain RIK151 (▪) and its ssExo-deficient derivatives DE1088 (RecB* RecD⁻ RecJ⁻) (▴), DE1089 (RecB* RecD⁻ RecJ⁻ ExoI⁻) (•), and DE1090 (RecB* RecD⁻ RecJ⁻ ExoVII⁻) (▾); DE101 (♦) is a recB-deficient mutant. Fraction survival is given as a fraction of unirradiated culture. Each value is a mean of two independent experiments.

Figure 2.

Model for transferred donor DNA (depicted by thick lines) processing in recD and wild-type Hfr transconjugants (A) and P1 transductants (B). Wavy and thin lines represent F plasmid DNA and recipient chromosome, respectively. RecA, RecBC, and RecBCD proteins are shown as a circle, triangle, and rectangle, respectively. Details of the reactions at each stage are described in the text.