Method for the isolation of Escherichia coli mutants with enhanced recombination between chromosomal duplications

SummaryThe Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo. We have combined the K72R variant of RecA with another mutation, RecA E38K (recA730). In vitro, the double mutant RecA E38K/K72R (recA730,2201) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/ K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro.

Section: Strain Constructionmentioning

confidence: 99%

RecA‐mediated SOS induction requires an extended filament conformation but no ATP hydrolysis

Gruenig

Renzette

Long

et al. 2008

“…Another common feature of recombination-dependent mutants in E. coli, such as polA, lig, dam or rdgB, is that they are hyper-rec, apparently because they use recombination for repair more frequently than wild-type cells do (Marinus and Konrad, 1976;Konrad, 1977;Zieg et al, 1978;Clyman and Cunningham, 1987). Interestingly, dut mutants, deficient in deoxyuridine triphosphatase (Hochhauser and Weiss, 1978), are also hyper-rec (Konrad and Lehman, 1975) and have elevated levels of single-strand interruptions in their DNA due to the excision of incorporated uracils by uracil DNA glycosylase (Ung) (Tye and Lehman, 1977), yet were reported to be recA-independent (Konrad and Lehman, 1975).…”

Section: Common Traits Of Rec-dependent Mutantsmentioning

confidence: 99%

RdgB acts to avoid chromosome fragmentation in Escherichia coli

Bradshaw

Kuzminov²

2003

125

SummaryBacterial RecA protein is required for repair of twostrand DNA lesions that disable whole chromosomes. recA mutants are viable, suggesting a considerable cellular capacity to avoid these chromosomedisabling lesions. recA -dependent mutants reveal chromosomal lesion avoidance pathways. Here we characterize one such mutant, rdgB / yggV , deficient in a putative inosine/xanthosine triphosphatase, conserved throughout kingdoms of life. The rdgB recA lethality is suppressed by inactivation of endonuclease V (gp nfi ) specific for DNA-hypoxanthines/ xanthines, suggesting that RdgB either intercepts improper DNA precursors dITP/dXTP or works downstream of EndoV in excision repair of incorporated hypoxathines/xanthines. We find that DNA isolated from rdgB mutants contains EndoV-recognizable modifications, whereas DNA from nfi mutants does not, substantiating the dITP/dXTP interception by RdgB. rdgB recBC cells are inviable, whereas rdgB recF cells are healthy, suggesting that chromosomes in rdgB mutants suffer double-strand breaks. Chromosomal fragmentation is indeed observed in rdgB recBC mutants and is suppressed in rdgB recBC nfi mutants. Thus, one way to avoid chromosomal lesions is to prevent hypoxanthine/xanthine incorporation into DNA via interception of dITP/dXTP.

“…The hyper-recombinicity of an E. coli strain is usually assessed using the Konrad assay, which detects restoration of a wild-type tacZ gene in one of two inversely oriented /acZ sequences cari7ing non-overlapping deletions (Konrad, 1977). This restoration requires RecA and RecBCD proteins and is independent of RecF (Zieg and Kushner, 1977), which is indicative of DSB repair, ttg, poiA, and dam mutants of E, coti are all hyper-rec in the Konrad assay and also exhibit increased recombination between the F' plasmid and a chromosome (Marinus and Konrad, 1976;Konrad, 1977;Bale etai, 1979).…”

Section: Increased Number Of Single-strand Interruptions In Dna Stimumentioning

confidence: 99%

Collapse and repair of replication forks in Escherichia coli

Kuzminov

1995

389

384

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.