DNA Repair Pathways

• Genome of the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans Viewed from the Perspective of Comparative Genomics

D. radiodurans has repair pathways that include excision repair, mismatch repair, and recombinational repair. Generally, no marked error-prone SOS response is observed in D. radiodurans. However, there have been a few reports consistent with SOS response, where preexposure to low doses of ionizing radiation, UV, or hydrogen peroxide causes a low level of subsequent increased resistance to DNA damage (twofold or less). Since the SOS response is not always mutagenic, the absence of DNA damage-induced mutagenesis observed in D. radiodurans cannot be taken as evidence against the existence of the SOS response in this bacterium. Photoreactivation is not present, and it has been reported that the adaptive response to alkylation damage is also absent. It is known that following DNA damage, there are changes in the cellular abundance of proteins, with enhanced synthesis of four to nine proteins, as judged by sodium dodecyl sulfate-polyacrylamide protein gels. Included in this group of proteins are probably RecA, elongation factor Tu, and KatA. While there are many predicted DNA repair genes and pathways in the D. radioduransgenome, only a few of its DNA repair enzymatic activities and/or genes have been evaluated for their biochemical activities. The UvrA protein and its gene have been detected, and it has been identified as a component of nucleotide excision repair. UV endonuclease-beta has been purified and found to be a 36-kDa manganese-requiring protein, which is thus far only known to recognize UV-induced pyrimidine cyclobutane dimers, incising them as an endonuclease rather than as a glycosylase. Other repair-related activities detected in extracts of D. radiodurans include uracil DNA glycosylase, a thymine glycol glycosylase, and a deoxyribophosphodiesterase. DNA polymerase I activity is present and is necessary for resistance to both UV and ionizing radiation. Both UvrA and DNA polymerase I deficiencies can be fully complemented by the expression of E. coli UvrA and DNA polymerase I proteins in D. radiodurans mutants, respectively. However, this is not the case for D. radiodurans recA, which appears to play a more important role in the extreme radiation resistance phenotype.

Genomic analyses show that the number of genes identified in D. radiodurans that are known to be involved in DNA repair is less than reported for E. coli or the highly sensitive bacterium S. oneidensis, and few novel genes involved in radiation resistance have been identified in D. radiodurans so far (Table 1).

The D. radiodurans RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the E. coli RecA protein. D. radiodurans recA mutants are highly sensitive to UV and ionizing radiation. Most of the amino acid residues that are distinct in Deinococcus and could be responsible for the structural and functional differences between the RecA proteins of Deinococcus and other bacteria are also present in the RecA sequence of Thermus thermophilus (Omelchenko et al., 2006). In this context, early work by Carroll et al (1996) reported that E. coli RecA did not complement an IR-sensitive D. radiodurans recA point-mutant (rec30) and that expression of D. radiodurans RecA in E. coli was lethal. More recently, however, it has been reported that E. coli recA can provide partial complementation to a D. radiodurans recA null mutant (Schlesinger, 2007). This suggests that the D. radiodurans RecA protein is not as unusual as initially believed, but rather is more analogous to polA anduvrA of D. radiodurans, which can be functionally replaced by E. coli orthologs.

D. radiodurans RecA has recently been purified and characterized. In vitro, it has been shown to catalyze the spectrum of activities classically attributed to RecA proteins: (i) it forms striated filaments on single-stranded DNA and double-stranded DNA; (ii) it promotes an efficient DNA strand exchange reaction; and (iii) it has a DNA-dependent nucleoside triphosphatase activity. However,D. radiodurans RecA is distinct from other well-characterized RecAs (e.g., from the gram-negative E. coli) in its nucleoside triphosphatase and DNA strand exchange activities. Unlike E. coli RecA, D. radiodurans RecA does not hydrolyze ATP at pH 7.5, although it exhibits some ATPase activity at lower pHs. In contrast, it is very effective at hydrolyzing dATP over a broad pH range.

The existence of a very efficient recA-independent single-stranded DNA annealing repair pathway has been reported for D. radiodurans. This pathway is active during and immediately after DNA damage and before the onset of recA-dependent repair. It can repair about one-third of the 150 to 200 DSBs per chromosome following exposure to 1.75 megarads. It has also been reported that unlike other organisms, D. radiodurans RecA is not present in the undamaged deinococcal cell but is synthesized only following DNA damage and following repair. D. radiodurans RecA is apparently expressed in D. radiodurans only following extreme DNA damage, and it is noteworthy that the recA-defective D. radiodurans strain rec30 is more radiation resistant than E. coli. It is possible that the greater resistance of rec30 arises from the presence of multiple copies of its genome in combination with the single-stranded DNA-annealing repair pathway, which is fully functional in this mutant. Together, this evidence supports the idea that D. radiodurans RecA is not necessary for the repair of nonextreme DNA damage (~10 DSB/chromosome, ~100 kilorads) and that D. radiodurans RecA may be activated only when DNA is highly damaged (>100 kilorads).

Replication, Repair, and Recombination

D. radiodurans contains all the typical bacterial genes that comprise the basal DNA replication machinery (Table 1). The number of paralogs and the domain organization of the DNA polymerase III -subunit is variable in the major bacterial divisions in terms of the presence of an active or inactivated PHP domain, which is predicted to possess phosphatase activity, and the proofreading 3'-5' exonuclease domain. D. radiodurans encodes a single-subunit that is most similar to proteobacterial polymerases and does not contain the 3'-5' exonuclease, which is encoded by a separate gene orthologous to E. coli dnaQ. Unlike the proteobacterial orthologs, however, the Deinococcus polymerase contains an apparently active PHP domain. This appears to represent the ancestral bacterial state of the replicative DNA polymerase, which is also seen in bacteria like Synechocystis and Aquifex. In addition to typical proteins involved in replication, Deinococcus encodes DNA polymerase X, which is similar to the eukaryotic DNA polymerase beta (references and and references therein), and is relatively uncommon in prokaryotes. Deinococcus polymerase X contains an N-terminal nucleotidyltransferase domain and a C-terminal PHP hydrolase domain, the same domain architecture that is seen in homologs from B. subtilis and Methanobacterium thermoautotrophicum; this conservation of domain organization suggests horizontal transfer of the polymerase X gene. Notably, along with a few other bacteria, such as Synechocystis and Aquifex,Deinococcus encodes three small nucleotidyltransferases (DR1806, DR0679, and DR0248), which are expanded in archaea. These "minimal" nucleotidyltransferases are typically accompanied by a small protein that is fused to the nucleotidyltransferase in the DR0248 protein; the function of this protein, however, has not been characterized directly but is likely to be coupled to that of the nucleotidyltransferases.

Bacterial DNA repair includes several partially redundant pathways and generally shows considerable flexibility. We investigated the predicted repair system components of D. radiodurans in detail, to detect any possible correlation with its exceptional radioresistant and desiccation-resistant phenotype. Generally, it appears that Deinococcus possesses a typical bacterial system for DNA repair and that, commensurate with the genome size, its repair pathways even appear to be less complex and diverse than those of bacteria with larger genomes, such as E. coli and B. subtilis. At the same time, there are several interesting and unusual aspects of the predicted layout of the repair systems in Deinococcus that may be linked to its phenotype (Table 1).

The nucleotide excision repair system that consists of the UvrABC excinuclease and the UvrD and Mfd (transcription-repair coupling factor) helicases is fully represented in D. radiodurans. Also present are the main components of the base excision repair system including several nucleotide glycosylases and endonucleases, namely, MutM (formamidopyrimidine and 8-oxoguanine DNA glycosylase); MutY (8-oxoguanine DNA glycosylase and apurinic DNA endonuclease-lyase); two paralogous uracil DNA glycosylases (Ung homologs); an additional, recently identified enzyme that has the same activity but is unrelated to Ung (DR1751); endonucleases III (Nth) and V (YjaF); and exonuclease III (XthA). Deinococcus lacks two key enzymes involved in the repair of UV-damaged DNA in other organisms, namely, endonuclease IV (AP-endonuclease) and photo-lyase. Instead, it encodes a typical bacterial UV endonuclease III (thymine glycol-DNA glycosylase) and, more unexpectedly, a TIM-barrel fold nuclease characteristic of eukaryotes and most closely related to the UV endonuclease of Neurospora. Eukaryotic-type topoisomerase IB is a truly unexpected protein to be identified in the Deinococcus genome and also could play a role in UV resistance.

The repertoire of recombinational repair genes in Deinococcus includes orthologs of most of the E. coli genes involved in this process (Table 1), but the RecBCD recombinase is missing. While this complex is not universal in bacteria, it is a major component of recombination systems in most free-living species. In Deinococcus, where recombination is thought to be an important contributor to damage-resistance, the absence of this ATP-dependent exonuclease is unexpected. Deinococcus does encode an apparent ortholog of one of the helicase-related subunits of this complex, RecD, but not the other subunits. The RecD protein in Deinococcusis unusual in that it contains an N-terminal region of about 200 amino acid residues that consist of three tandem predicted HhH DNA-binding domains; this unusual domain organization of the RecD protein is shared with B. subtilis and Chlamydia. Such dissociation of RecD from the RecB and RecC subunits is not unique to Deinococcus; "solo" RecD-related proteins are also present inM. jannaschii and in yeast. The function(s) of RecD, once outside the recombinase complex, is unknown.

Another component of the recombinational repair system in Deinococcus that has an unusual domain architecture is the RecQ helicase. It contains three tandem copies of the C-terminal helicase-RNase D (HRD) domain, instead of the single copy present in all other bacteria except Neisseria that similarly possesses three copies. RecQ sequences from Neisseria and Deinococcus are more similar to each other than to any other homologs, which, together with the distinctive triplication of the HRD domain, indicates that the recQ gene has been exchanged between bacteria from these two distant lineages. In addition, Deinococcus encodes a protein (DR2444) that contains an HRD domain and a domain homologous to cystathionine gamma-lyase; this is the first example of an HRD domain that is not associated with either a helicase or a nuclease (although it is possible that the domain organization of this protein is an artifact caused by a frameshift). This propagation of the HRD domain in Deinococcus could contribute to the repair phenotype given the interactions of RecQ with RecA in recombination.

The methylation-dependent mismatch repair system of D. radiodurans includes the MutS and MutL ATPases and endonuclease VII (XseA). Orthologs of the site-specific methylases Dcm and Dam, which are associated with mismatch repair, are not readily detectable. It appears likely, however, that other distantly related DNA methylases predicted in D. radiodurans could perform similar functions.

Like other bacteria with large genomes, D. radiodurans encodes the LexA repressor-autoprotease (DRA0344), which in E. coli andB. subtilis controls the expression of the SOS regulon. In addition, unlike any of the other bacterial genomes studied, D. radiodurans encodes a second, diverged copy of LexA (DRA0074), which retains the same arrangement of the helix-turn-helix (HTH) DNA-binding domain and the autoprotease domain. Attempts to identify LexA-binding sites and the composition of the putative SOS regulon in D. radiodurans have been unsuccessful (M. S. Gelfand, personal communication). This suggests that D. radiodurans does not possess a functional SOS response system, which is in agreement with the results of previous experimental studies. Furthermore, Deinococcus does not encode proteins of the DinP/UmuC family, nonprocessive DNA polymerases that play a critical role in translesion DNA synthesis and associated error-prone repair such as SOS repair in E. coli.

In addition to orthologs of well-characterized repair proteins, Deinococcus encodes several unusual proteins and expanded protein families that are less confidently associated with repair but might contribute to the unusual effectiveness of the repair and recombination systems in this bacterium.

Table 1. Genes coding for replication, repair, and recombination functions in D. radioduransa

From: Makarova et al., Microbiology and Molecular Biology Reviews (2001) Vol. 65, No. 1, 44-79