Juan Carlos Gómez Alonso

The Bacillus subtilis chromatin‐associated protein Hbsu is involved in DNA repair and recombination

The Bacillus subtilis hbs gene encodes an essential chromatin‐associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non‐specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site‐directed mutants in the hbs gene and used these mutant genes to substitute the wild‐type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe‐47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg‐55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and (i protein‐mediated site‐specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recti and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA‐damaging agents methylmethane sulphonate (MMS) or 4‐nitroquinoline‐1‐oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20‐ to 50‐fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.

Homologous recombination in low dC + dG Gram-positive bacteria

Homologous recombination is a process involved in the maintenance of chromosome integrity, in shaping the evolution of pathogens, in the resistance to antibiotic treatment, and profoundly affecting evolution. In low dC + dG Gram-positive bacteria genetic recombination of a non-replicative homologous DNA, which enters into the cell via transduction or conjugation, proceeds mainly by the double-strand break repair machinery, and this process can be limited by the host restriction system. However, transformation is not limited by sequence divergence (up to 17%), with the activity of restriction endonucleases, or the mismatch repair system only marginally affecting it. The transforming non-replicative homologous DNA, which has a single-stranded nature, is paired with recipient DNA by RecA protein, with the help of redundant accessory proteins, and the intermediates are resolved by a D-loop resolvase. If the transforming DNA does not share homology with the recipient (plasmid establishment), DNA replication and certain recombination proteins other than RecA are required.