Organisms like Dictyostelium discoideum, often referred to as DNA damage “extremophiles”, can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other “extremophiles” can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA), translesion synthesis (TLS), and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage–resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents.

https://www.mrc-lmb.cam.ac.uk/genomes/mkayikci/MendeleyMadanPapers/2009_PLoS%20genetics_Zhang%20et%20al._Xpf%20and%20not%20the%20Fanconi%20anaemia%20proteins%20or%20Rev3%20accounts%20for%20the%20extreme%20resistance%20to%20cisplatin%20in%20Dic.pdf

Xpf and not the Fanconi anaemia proteins or Rev3 accounts for the extreme resistance to cisplatin in Dictyostelium discoideum.

Emerging models for DNA repair: Dictyostelium discoideum as a model for nonhomologous end-joining

Oxidative stress may play an important role in the pathogenesis of age-related macular degeneration (AMD). Mitochondria produce reactive oxygen species (ROS), which induce degenerative changes typical for AMD. Mitochondrial DNA (mtDNA) is targeted by ROS and it is considered to be more vulnerable to damage than nuclear DNA (nDNA) due to the impaired DNA repair system, lack of nucleosomal organization and close vicinity of mitochondrial oxidative chain. Some reports suggest the association between mtDNA damage and AMD. However, the metabolism of mtDNA is mainly determined by the expression of nDNA. Therefore, the extent of damage to mtDNA in retinal cells depends on the overall efficacy of nDNA repair, which decreases with age. We showed an association between nDNA damage and repair and AMD. Also well-recognized factors of AMD pathogenesis, age and smoking, may exert their effects through the DNA damage and repair. In conclusion, DNA damage and repair, both in mitochondrial and nuclear genome, may play an important role in the pathogenesis of AMD, and their mutual relationship in this disease needs further study.

DNA damage and repair in age-related macular degeneration.

Recombinase enzymes promote DNA repair by homologous recombination. The genes that encode them are ancestral to life, occurring in all known dominions: viruses, Eubacteria, Archaea and Eukaryota. Bacterial recombinases are also present in viruses and eukaryotic groups (supergroups), presumably via ancestral events of lateral gene transfer. The eukaryotic recA genes have two distinct origins (mitochondrial and plastidial), whose acquisition by eukaryotes was possible via primary (bacteria–eukaryote) and/or secondary (eukaryote–eukaryote) endosymbiotic gene transfers (EGTs). Here we present a comprehensive phylogenetic analysis of the recA genealogy, with substantially increased taxonomic sampling in the bacteria, viruses, eukaryotes and a special focus on the key eukaryotic supergroup Amoebozoa, earlier represented only by Dictyostelium . We demonstrate that several major eukaryotic lineages have lost the bacterial recombinases (including Opisthokonta and Excavata), whereas others have retained them (Amoebozoa, Archaeplastida and the SAR-supergroups). When absent, the bacterial recA homologues may have been lost entirely (secondary loss of canonical mitochondria) or replaced by other eukaryotic recombinases. RecA proteins have a transit peptide for organellar import, where they act. The reconstruction of the RecA phylogeny with its EGT events presented here retells the intertwined evolutionary history of eukaryotes and bacteria, while further illuminating the events of endosymbiosis in eukaryotes by expanding the collection of widespread genes that provide insight to this deep history.

https://royalsocietypublishing.org/doi/pdf/10.1098/rspb.2016.1453

Evolution of bacterial recombinase A (recA) in eukaryotes explained by addition of genomic data of key microbial lineages.

Title of Dissertation: BIOLOGICAL CHARACTERIZATION OF RECD MUTANTS IN DEINOCOCCUS RADIODURANS Matthew David Servinsky, Doctor of Philosophy, 2008 Dissertation directed by: Professor Douglas A. Julin Department of Chemistry and Biochemistry The RecD protein in E. coli and other gram negative bacteria functions as a subunit in concert with RecB and RecC to form the heterotrimeric RecBCD complex. It is involved in degrading foreign linear DNA, providing a block against viral invaders, and has been shown to inhibit recombination. This complex also catalyzes the first step in the highly regulated enzymatic process of DSBR via homologous recombination (HR). DSBR via HR is thought to be the major route for processing double strand breaks in E. coli. The first step is known as initiation and generates the substrate for the next enzyme in the pathway, RecA. In contrast to E. coli, most gram positive bacteria, use a set of genes known as the AddAB genes to accomplish this same task, to produce the substrate for RecA. The gram positive bacterium Deinococcus radiodurans is remarkable in its ability to withstand DNA damaging agents such as MMC, gamma irradiation, hydrogen peroxide and UV irradiation. While D. radiodurans has a RecA homolog that has been shown to be critical for repair of double strand breaks, sequence analysis has not revealed homologs of AddAB or RecBC. A sequence with high similarity to recD was found. D. radiodurans RecD has an extended N-terminus grouping it with a sub-family of recD-like genes. The gene was expressed in E. coli and purified. Its biochemical activities were published by Wang and Julin, 2004, showing it had 5’-3’ helicase activity like RecD proteins from other organisms. In this study a recD mutation in D. radiodurans was generated. The mutant was sensitive to gamma and UV irradiation, and hydrogen peroxide. The mutant was insensitive to MMC and MMS. Additionally the mutant showed an increased capacity to be transformed by exogenous DNA. These results imply a role in DNA repair through some form of recombinational repair. This finding is the first published result of a member of the recD-like sub-family of proteins that suggests role in DNA repair. BIOLOGICAL CHARACTERIZATION OF RECD MUTANTS IN DEINOCOCCUS RADIODURANS

Biological characterization of RecD mutants in Deinococcus radiodurans

A homolog of Escherichia coli RecA in mitochondria of the cellular slime mold Dictyostelium discoideum.

Reactive oxygen species (ROS) are significant source of cellular and DNA damage. Organisms express various enzymes, such as superoxide dismutases (SODs), that detoxify ROS in order to protect themselves. SODs convert superoxide anions to H2O2, which is then disproportionated to water by catalases or peroxidases. SODs are divided into four subfamilies according to cofactors: copper-/zinc-containing SOD (Cu/ZnSOD), manganese-containing SOD (MnSOD), iron-containing SOD (FeSOD) and nickel-containing SOD (NiSOD) (Fridovich, 1995; Miller, 2004). Because of their important role, SODs are conserved widely in prokaryotes and eukaryotes. The social amoeba Dictyostelium discoideum, which feeds on bacteria in soil, is highly resistant to oxidative stress (Garcia et al., 2000). Thus, D. discoideum is thought to be a system suitable for studying mechanisms that function in protection of organisms from ROS. Four Cu/ZnSODs have been identified from D. discoideum. They were designated SodA (Garcia et al., 2000), SodB (Tsuji et al., 2002), SodC (Tsuji et al., 2003) and SodD (Akaza et al., 2002). In addition, four proteins that are predicted to be SODs can be found in D. dis21 Jpn. J. Protozool. Vol. 39, No. 1. (2006)

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Identification of a gene coding for manganese superoxide dismutase from the cellular slime mold Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum, a lower eukaryote feeding on bacteria in soil, is highly resistant to DNA damaging agents. D. discoideum expresses at least two RecA homologs, Rad51 and mitochondria localizing RecA. Rad51 consisting of 351 amino acid residues is 69% identical to human Rad51. UV light-irradiation and H2O2-treatment increased the expression level of Rad51, suggesting that Rad51 is required for DNA repair in D. discoideum. A gene disruption mutant was not viable, suggesting that Rad51 is essential for D. discoideum.

Analysis of Rad51 in the Social Amoeba Dictyostelium Discoideum : Sequence, Induction and Disruption

In the cellular slime mold Dictyostelium discoideum, Countin2 and Countin3 proteins are thought to limit the minimum size of the multicellular structure since countin2- and countin3- strains form small fruiting bodies. Expression levels of the cell-cell adhesion proteins, gp24 and gp80, in wild type and null mutants were compared. During the process of aggregation, countin2- cells expressed lower levels of gp24 and gp80 than those expressed by the wild type. The expression levels of gp24 and gp80 in countin3- cells were almost the same as those in wild-type cells in the early stages of aggregation but were lower than wild-type levels in the late stages. These results indicate that Countin2 and Countin3 proteins enhance the expression of gp24 and gp80 in D. discoideum in different ways.

Yasuna Hasegawa

Papers

A homolog of Escherichia coli RecA in mitochondria of the cellular slime mold Dictyostelium discoideum.

Identification of a gene coding for manganese superoxide dismutase from the cellular slime mold Dictyostelium discoideum

Analysis of Rad51 in the Social Amoeba Dictyostelium Discoideum : Sequence, Induction and Disruption

Expression of Genes Coding for Cell-Cell Adhesion Proteins in Dictyostelium discoideum Mutants that Form Small Fruiting Bodies

The recA-deficient Dictyostelium discoideum Forms Large Fruiting Bodies