Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). The various causes of double-strand breaks (DSBs) result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, DNA polymerases, and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during variable (diversity) joining [V(D)J] recombination and class switch recombination (CSR). Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation (IR), but also severely immunodeficient.

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The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents. Spore resistance to wet heat is determined largely by the water content of spore core, which is much lower than that in the growing cell protoplast. A lower core water content generally gives more wet heat-resistant spores. The level and type of spore core mineral ions and the intrinsic stability of total spore proteins also play a role in spore wet heat resistance, and the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP) protects DNA against wet heat damage. However, how wet heat kills spores is not clear, although it is not through DNA damage. The alpha/beta-type SASP are also important in spore resistance to dry heat, as is DNA repair in spore outgrowth, as Bacillus subtilis spores are killed by dry heat via DNA damage. Both UV and gamma-radiation also kill spores via DNA damage. The mechanism of spore resistance to gamma-radiation is not well understood, although the alpha/beta-type SASP are not involved. In contrast, spore UV resistance is due largely to an alteration in spore DNA photochemistry caused by the binding of alpha/beta-type SASP to the DNA, and to a lesser extent to the photosensitizing action of the spore core's large pool of dipicolinic acid. UV irradiation of spores at 254 nm does not generate the cyclobutane dimers (CPDs) and (6-4)-photoproducts (64PPs) formed between adjacent pyrimidines in growing cells, but rather a thymidyl-thymidine adduct termed spore photoproduct (SP). While SP is formed in spores with approximately the same quantum efficiency as that for generation of CPDs and 64PPs in growing cells, SP is repaired rapidly and efficiently in spore outgrowth by a number of repair systems, at least one of which is specific for SP. Some chemicals (e.g. nitrous acid, formaldehyde) again kill spores by DNA damage, while others, in particular oxidizing agents, appear to damage the spore's inner membrane so that this membrane ruptures upon spore germination and outgrowth. There are also other agents such as glutaraldehyde for which the mechanism of spore killing is unclear. Factors important in spore chemical resistance vary with the chemical, but include: (i) the spore coat proteins that likely react with and detoxify chemical agents; (ii) the relative impermeability of the spore's inner membrane that restricts access of exogenous chemicals to the spore core; (iii) the protection of spore DNA by its saturation with alpha/beta-type SASP; and (iv) DNA repair for agents that kill spores via DNA damage. Given the importance of the killing of spores of Bacillus species in the food and medical products industry, a deeper understanding of the mechanisms of spore resistance and killing may lead to improved methods for spore destruction.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2005.02736.x

Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals

Orthologs and paralogs are two fundamentally different types of homologous genes that evolved, respectively, by vertical descent from a single ancestral gene and by duplication. Orthology and paralogy are key concepts of evolutionary genomics. A clear distinction between orthologs and paralogs is critical for the construction of a robust evolutionary classification of genes and reliable functional annotation of newly sequenced genomes. Genome comparisons show that orthologous relationships with genes from taxonomically distant species can be established for the majority of the genes from each sequenced genome. This review examines in depth the definitions and subtypes of orthologs and paralogs, outlines the principal methodological approaches employed for identification of orthology and paralogy, and considers evolutionary and functional implications of these concepts.

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Orthologs, Paralogs, and Evolutionary Genomics 1

Relatively little is known about the biochemical basis of the capacity of Deinococcus radiodurans to endure the genetic insult that results from exposure to ionizing radiation and can include hundreds of DNA double-strand breaks. However, recent reports indicate that this species compensates for extensive DNA damage through adaptations that allow cells to avoid the potentially detrimental effects of DNA strand breaks. It seems that D. radiodurans uses mechanisms that limit DNA degradation and that restrict the diffusion of DNA fragments that are produced following irradiation, to preserve genetic integrity. These mechanisms also increase the efficiency of the DNA-repair proteins.

Deinococcus radiodurans - the consummate survivor.

In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis. These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.

https://science.sciencemag.org/content/sci/297/5587/1686.full.pdf

Identification of a DNA Nonhomologous End-Joining Complex in Bacteria

In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.

https://science.sciencemag.org/content/sci/306/5696/683.full.pdf

Mycobacterial Ku and Ligase Proteins Constitute a Two-Component NHEJ Repair Machine

The present invention relates to the cloning and characterisation of a prokaryotic DNA repair ligase, which is shown to possess a range of activities that allow the ligation and repair of non-compatible DNA ends and double strand breaks (DSBs). The enzyme has a range of applications in the manipulation and cloning of nucleic acids.

Marina Della

Papers

Identification of a DNA Nonhomologous End-Joining Complex in Bacteria

Mycobacterial Ku and Ligase Proteins Constitute a Two-Component NHEJ Repair Machine

Prokaryotic dna repair ligases