The genetic dependence of RecBCD-Gam mediated double strand end repair in Escherichia coli.

The repair of double strand breaks after gamma-irradiation in wild-type Escherichia coli lysogenic for lambda cI857 red3 is more efficient when lambda Gam protein is present. This phenomenon, called gam dependent radioresistance, requires the interaction of RecBCD enzyme and Gam protein. We compared cell survival after gamma-irradiation in wild-type and mutant lysogens with and without induction of Gam by transient heat treatment of the cells (6 min, 42 degrees C). The main conclusions are: (1) the RecBCD-Gam pathway of recombination repair is similar but not equivalent to RecBCD, a pathway operating in recD mutants; (2) the RecBCD-Gam pathway is dependent on recJ, recQ and recN gene products and it is proposed that the RecBCD-Gam complex has ability to load RecA protein onto single strand DNA.

In vivo studies of the Escherichia coli RecB polypeptide lacking its nuclease center.

In vitro, RecB1-929, the truncated Escherichia coli RecB polypeptide, comprising the N-terminal (helicase) domain of RecB, can combine with RecC and RecD subunits of RecBCD enzyme. The resulting RecB1-929CD heterotrimer is a potent helicase; due to the loss of the nuclease center of RecB, it is devoid of DNase activities. By making use of the RecB1-929-producing plasmid pMY100, the in vivo behavior of this truncated polypeptide was studied. The following observations were made. (i) Large amounts of RecB1-929 in the pulse-heated lambdacI857gam+ lysogens prevented the growth of a gene 2 mutant of bacteriophage T4. It may be inferred that lambda-Gam protein, which otherwise inhibits RecBCD DNase and thus permits the growth of this phage, is bound by the helicase domain of RecB. (ii) The simultaneous presence of RecB1-929, RecC, and RecD did not restore recombination proficiency and ultraviolet resistance of recB cells. (iii) The presence of RecB1-929 did not alter recombination and repair processes in wild-type (recBCD+) cells. Even excessively large amounts of this truncated polypeptide did not reduce degradation of chromosomal DNA damaged by y-rays. It may be inferred that under in vivo conditions, the 30-kDa domain of RecB is essential for assembly of the RecBCD enzyme and/or for holding its three subunits together.

Overproduction of the RecD polypeptide sensitizes Escherichia coli cells to gamma-radiation.

We investigated DNA metabolism in Escherichia coli cells carrying the multicopy recD+ plasmid (pKI13). In the presence of pKI13, the cellular level of the recD gene product (RecD polypeptide) is amplified at least 60-fold. Overproduction of the RecD polypeptide has no effect on UV repair and conjugational recombination. In contrast, high cellular levels of this polypeptide sensitize wild-type cells to gamma-radiation; also, they increase the rate of radiation-induced DNA degradation. A possible mechanism for the enhancement of gamma-ray-induced killing by large amounts of the RecD polypeptide is discussed.

Interaction of RecBCD enzyme with DNA damaged by gamma radiation.

The DNA of a gene 2 mutant (T4 2-) of phage T4 is degraded by RecBCD enzyme in the bacterial cytoplasm. Under normal conditions, recBCD+ cells are therefore incapable of supporting the growth of phage T4 2-. Only if the nucleolytic activity of RecBCD enzyme is absent from the cytoplasm are T4 2(-)-infected bacteria able to form plaques. We found that recBCD+ cells can form plaques if, before infection with T4 2-, they have been exposed to gamma radiation. It is suggested that gamma ray-induced lesions of the bacterial DNA (e.g., double-strand breaks) bind RecBCD enzyme. This binding enables the enzyme to begin to degrade the bacterial chromosome, but simultaneously prevents its degradative action on the ends of minor DNA species, such as unprotected infecting phage chromosomes. Degradation of the chromosomal DNA, which occurs during the early postirradiation period, ceases about 60 min after gamma ray exposure. The reappearance of the nucleolytic action of RecBCD enzyme on T4 2- DNA accompanies the cessation of degradation of bacterial DNA. Both, this cessation and the reappearance of the nucleolytic action of ReCBCD enzyme on T4 2- DNA depend on a functional recA gene product. These results suggest that postirradiation DNA degradation is controlled by the recA-dependent removal of RecBCD enzyme from the damaged chromosome. By making use of the temperature-sensitive mutant recB270, we showed that RecBCD-mediated repair of gamma ray-induced lesions occurs during the early postirradiation period, i.e. during postirradiation DNA degradation. It is shown that the RecD subunit of RecBCD enzyme also participates in this repair.

A possible interaction of single-strand binding protein and RecA protein during post-ultraviolet DNA synthesis.

The mechanism of DNA replication in ultraviolet (UV)-irradiated Escherichia coli is proposed. Immediately after UV exposure, the replisome aided by single-strand DNA-binding protein (SSB) can proceed past UV-induced pyrimidine dimers without insertion of nucleotides. Polymerisation eventually resumes somewhere downstream of the dimer sites. Due to the limited supply of SSB, only a few dimers can be bypassed in this way. Nevertheless, this early DNA synthesis is of great biological importance because it generates single-stranded DNA regions. Single-stranded DNA can bind and activate RecA protein, thus leading to induction of the SOS response. During the SOS response, the cellular level of RecA protein increases dramatically. Due to the simultaneous increase in the concentration of ATP, RecA protein achieves the high-affinity state for single-stranded DNA. Therefore it is able to displace DNA-bound SSB. The cycling of SSB on and off DNA enables the replisome to bypass a large number of dimers at late post-UV times. During this late replication, the stoichiometric amounts of RecA protein needed for recombination are involved in the process of postreplication repair.

The recB gene product is essential for exonuclease V-dependent DNA degradation in vivo.

The recB21 mutation abolishes the exonuclease activity of the RecBCD enzyme (exonuclease V) of Escherichia coli. This might be due to the polar effect of recB21 on expression of the recD gene, the product of which is an essential component of the RecBCD enzyme. To achieve synthesis of the recD gene product, the recD+ plasmid was introduced into the recB21 mutant. Degradation of the endogenous DNA damaged by gamma-rays and degradation of the DNA of a phage T4 gene 2 mutant were nevertheless abnormally small in this strain. Thus, the functional recB gene product is required for the degradative function of the RecBCD enzyme.

Post-ultraviolet DNA synthesis in the absence of repair: role of the single-strand DNA-binding protein.

Post-ultraviolet DNA synthesis kinetics were investigated in the Escherichia coli uvrA recA strain and its isogenic counterpart, overproducing single-strand DNA-binding protein (SSB). It was demonstrated that large quantities of SSB enhance the capacity of the unmodified replisome to use the UV-damaged template for DNA synthesis. DNA thus synthesized is of low molecular weight, as shown by sedimentation in alkaline sucrose gradients. It is therefore suggested that SSB actively participates in the replisome translocation past dimers and/or the initiation of new DNA chains downstream of these lesions.