RESUMEN
BACKGROUND: In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double-stranded (ds) DNA exonuclease/helicase. Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. ChiEc is frequent and over-represented on its genome, which is thought to be related to its role in dsDNA break repair. We previously identified a Chi-like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram-positive bacterium Lactococcus lactis. ChiLl and RexAB are functional analogues of ChiEc and RecBCD. RESULTS: We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. Analysis of ChiLl distribution on the L. lactis chromosome reveals that ChiLl is frequent, highly over-represented, and oriented with respect to the direction of replication. CONCLUSION: Our results show that a single orientation of ChiLl interacts with RexAB. The active orientation is preferentially found on the replication leading strand of the L. lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork. We propose that orientation-dependence of Chi activity and over-representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase-Chi couples. Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.
Asunto(s)
Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Lactococcus lactis/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Inversión Cromosómica , Cromosomas Bacterianos/genética , Replicación del ADN , Exodesoxirribonucleasa V , Frecuencia de los Genes , Lactococcus lactis/metabolismo , Recombinación GenéticaRESUMEN
The availability of full genome sequences provides the bases for analyzing global properties of the genetic text. For example, oligonucleotide sequences that are over- or underrepresented can be identified by taking into account the overall genome composition and organization. One of the most overrepresented oligonucleotides in Escherichia coli is the Chi site, an octanucleotide that stimulates DNA repair by homologous recombination. Here we analyze the genomic distribution of Chi in E. coli and in the three other bacteria where a Chi sequence has been identified; note that Chi is a different sequence in each organism. For each bacterial genome, Chi sequences are frequent, regularly distributed, and overrepresented. This suggests that selection for Chi may have occurred during evolution to favor efficient repair of a damaged chromosome. Other characteristics of Chi distribution are not conserved and might reflect specific features of DNA repair in each host. The different sequence and characteristics of Chi in each microorganism suggest that selection for Chi occurred independently in different bacteria.
Asunto(s)
Bacterias/genética , Genoma Bacteriano , Bacillus subtilis/genética , Bacterias/enzimología , Secuencia de Bases , ADN Helicasas/genética , ADN Bacteriano/genética , Escherichia coli/genética , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Haemophilus influenzae/genética , Lactococcus lactis/genética , Datos de Secuencia Molecular , Recombinación Genética/genética , Análisis de SecuenciaRESUMEN
Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Secuencia de Bases , Replicación del ADN/genética , Regulación hacia Abajo , Escherichia coli/enzimología , Escherichia coli/genética , Exodesoxirribonucleasa V , Genes Bacterianos , Genoma Bacteriano , Recombinación GenéticaRESUMEN
The Escherichia coli Chi site 5'-GCTGGTGG-3' modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E. coli genome. Chi is also present much more frequently than predicted statistically for a random 8-mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae. The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5'-GNTGGTGG-3' and 5'-G(G/C)TGGAGG-3', showed Chi activity. As in E. coli, the H. influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over-represented, this feature is less marked than that of the E. coli Chi site. In contrast to E. coli, the H. influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.
Asunto(s)
ADN Helicasas/metabolismo , ADN Bacteriano/metabolismo , Escherichia coli/genética , Exodesoxirribonucleasas/metabolismo , Haemophilus influenzae/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencia de Bases , Recuento de Colonia Microbiana , Secuencia de Consenso , ADN Helicasas/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Genoma Bacteriano , Haemophilus influenzae/metabolismo , Peso Molecular , Oligonucleótidos/biosíntesis , Plásmidos , Recombinación Genética , Análisis de Secuencia , Especificidad por Sustrato , Transformación BacterianaRESUMEN
MOTIVATION: We created Micado, a database for managing genomic information, as part of the Bacillus subtilis genome programs. Its content will be progressively extended to the whole microbial world. RESULTS: A relational schema is defined for selective queries. It links eubacterial and archaeal sequences, genetic maps for Bacillus subtilis and Escherichia coli, and information on mutants. The latter comes from a new functional analysis project of unknown genes in B subtilis, and the database allows the community to curate information. To help queries from users, a graphical interface is built on SQL access to the database and provided through the WWW. We have automated imports of microbial sequences, and E. coli genetic map, by programming parsers of flat file distributions. These ensure smooth updates from molecular biology repositories on the Internet. Hyperlinks are created as a complement, to reference other general and specialized related information resources.