Wild-type RecA Research Articles

Isolation of altered recA polypeptides and interaction with ATP and DNA.

In this paper we describe the partial proteolytic digestion of recA proteins from Escherichia coli and Proteus mirabilis and the production and isolation of truncated recA polypeptides. A proteolytic fragment of the P. mirabilis recA protein bound single-strand DNA and ATP normally but has altered duplex DNA binding properties. This protein was shown to initiate but not complete DNA strand transfer from a DNA duplex to a complementary single strand. The product of the E. coli recA1 allele bound but could not hydrolyze ATP and the protein bound single-strand but not double-strand DNA. This protein did not appear to initiate the transfer of a strand from a linear duplex to a single-strand circle and inhibited the wild-type recA protein from performing strand transfer. We report that recA protein binds linear duplex DNA in a manner that enhances the rate of ligation by T4 DNA ligase. When heterologous single-strand DNA was added in addition to the duplex DNA large stable aggregates of protein and DNA were formed that could easily be sedimented from solution.

Defective complex formation between single-stranded DNA and mutant recA430 or recA1 protein of Escherichia coli.

The formation of a complex between recA protein and single-stranded DNA (ssDNA) was studied by filter-binding assays and sucrose density gradient centrifugation. In the presence of excess ATP, the wild-type recA protein formed salt-resistant complexes with ssDNA. One mutant recA430 (lexB30) protein bound to ssDNA slightly less effectively than wild-type protein, and the complexes had lost the stability to salt. Another mutant recA1 protein did not form complexes with ssDNA. On the other hand, in the absence of ATP, all proteins bound to ssDNA with the same efficiency, but all of the complexes were unstable in the presence of salt. The hybridization reaction in which homologous ssDNA is converted to double-stranded DNA (dsDNA) catalyzed by the recA430 protein was more sensitive to salt than that catalyzed by the wild-type protein. No hybridization reaction was found with the recA1 protein.

Primary structure analysis of the mutant recA 441 and recA 430 proteins.

The mutant recA 441 and recA 430 proteins from Escherichia coli have single amino acid substitutions which distinguish each of them from the wild type recA protein. recA 441 mutants express SOS functions at 42 degrees C even without DNA damage whereas recA 430 mutants are totally unable to express SOS functions at any temperature. Both mutant recA proteins have altered protease functions. However, the amino acid substitutions in each protein are located at a considerable linear distance from one another in their respective polypeptide chains, the recA 441 protein having a lysine in place of a glutamic acid residue at position 68, while the recA 430 protein has a serine in place of a glycine at position 204.

Biologically active recombinant formed through DNA pairing by purified recA protein in vitro.

We have detected in vitro homologous recombination mediated by purified recA protein of Escherichia coli as a recombinant phage produced by using the DNA packaging system of phage lambda. When double-stranded DNA of phage lambda carrying amber mutations is incubated with double-stranded DNA carrying the wild-type genes in the presence of recA protein, Mg++ and ATP, and the DNA packaged, amber+ recombinant phage is produced at a high frequency. This reaction depends completely upon the function of the wild-type recA protein. After incubation of 32P-labeled linear DNA (Form III) with bromouracil-labeled circular DNA (Form I-Form II mixture) in the presence of recA protein, Mg++ and ATP, about 10% of the 32P-counts band at an intermediate density in CsCl equilibrium gradient. This fraction yields a high percentage of the recombinant phage after DNA packaging and shows the alpha-shaped and sigma-shaped joint molecules of linear and circular DNA under the electron microscope. Furthermore, we demonstrate that a non-homologous region inhibits the recombination reaction when it is between the marker concerned and the closer cos end. Our results indicate that recA protein acts directly in the initial step of recombination to join the homologous double-stranded DNA and that the resulting molecule can be matured into the recombinant DNA.

The recA-dependent spontaneous degradation of proximal F merogenotes in Escherichia coli.

Proximal F' elements of KLF-1 type are relatively stable in Escherichia coli recA recipients. In such merodiploids the transferability of F'-DNA and the plasmid determined fertility functions are expressed. When introduced into the wild type recA+ cells the F'-DNA is degraded and several classes of DNA molecules of molar mass about 66 Mg/mol and lower exist in the cell in 1-2 copies per bacterial chromosome. As was detected by complementation analysis, the chromosomal genes determining the host specificity for DNA (hsd) originally located on the F' element seem to be salvaged during the process of DNA degradation probably by recombination with the bacterial chromosome.

Induction of SOS functions: Regulation of proteolytic activity of E. coil RecA protein by interaction with DNA and nucleoside triphosphate

Damage to cellular DNA or interruption of chromosomal DNA synthesis leads to induction of the SOS functions in E. coli. The immediate agent of induction is the RecA protein, which proteolytically cleaves and inactivates repressors, leading to induction of genes they control. RecA protein modified by tif mutations allows expression of SOS functions in the absence of inducing treatments. We show here that tif-mutant RecA protein is more efficient than wild-type RecA protein in interacting with DNA and nucleoside triphosphate. This result suggests that formation of a complex with DNA and nucleoside triphosphate is the critical event that activates RecA protein to destroy repressors after SOS-inducing treatments, and that damage to cellular DNA promotes this reaction by providing single-stranded DNA or an active nucleoside triphosphate or both. Since dATP is the most effective nucleoside triphosphate in promoting repressor cleavage, we suggest that it is the natural cofactor of RecA protein in vivo.