Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.

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Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli.

Exposure of Escherichia coli to a variety of DNA-damaging agents results in the induction of the global ‘SOS response’. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA-regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5′-TACTG(TA)5CAGTA-3′. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA-regulated genes. These searches identified a total of 69 potential LexA-regulated genes/operons with a heterology index of < 15 and included all previously characterized LexA-regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage-inducible gene expression in a wild-type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non-inducible lexA(Ind−) cell. These experiments have allowed us to identify seven new LexA-regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA-regulated gene is discussed.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2000.01826.x

Identification of additional genes belonging to the LexA regulon in Escherichia coli

When duplex DNA is altered in almost any way (replicated, recombined, or repaired), single strands of DNA are usually intermediates, and single-stranded DNA binding (SSB) proteins are present. These proteins have often been described as inert, protective DNA coatings. Continuing research is demonstrating a far more complex role of SSB that includes the organization and/or mobilization of all aspects of DNA metabolism. Escherichia coli SSB is now known to interact with at least 14 other proteins that include key components of the elaborate systems involved in every aspect of DNA metabolism. Most, if not all, of these interactions are mediated by the amphipathic C-terminus of SSB. In this review, we summarize the extent of the eubacterial SSB interaction network, describe the energetics of interactions with SSB, and highlight the roles of SSB in the process of recombination. Similar themes to those highlighted in this review are evident in all biological systems.

/pdf/ssb-as-an-organizer-mobilizer-of-genome-maintenance-5cb8l0xn6s.pdf

SSB as an organizer/mobilizer of genome maintenance complexes

The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.

Gregg-Jolly La

Papers

Identification of High Affinity Binding Sites for LexA which Define New DNA Damage-inducible Genes in Escherichia coli