Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components

Hydrolytic deamination of DNA cytosine residues results in U/G mispairs, pre-mutagenic lesions threatening long-term genetic stability. Hence, DNA uracil repair is ubiquitous throughout all extant life forms and base excision repair, triggered by a uracil DNA glycosylase (UDG), is the mechanistic paradigm adopted, as it seems, by all bacteria and eukaryotes and a large fraction of archaea. However, members of the UDG superfamily of enzymes are absent from the extremely thermophilic archaeon Methanothermobacter thermautotrophicus ΔH. This organism, as a hitherto unique case, initiates repair by direct strand incision next to the DNA-U residue, a reaction catalyzed by the DNA uridine endonuclease Mth212, an ExoIII homologue. To elucidate the detailed mechanism, in particular to identify the molecular partners contributing to this repair process, we reconstituted DNA uracil repair in vitro from only four purified enzymes of M. thermautotrophicus ΔH. After incision at the 5′-side of a 2′-d-uridine residue by Mth212 DNA polymerase B (mthPolB) is able to take over the 3′-OH terminus and carry out repair synthesis generating a 5′-flap structure that is resolved by mthFEN, a 5′-flap endonuclease. Finally, DNA ligase seals the resulting nick. This defines mechanism and minimal enzymatic requirements of DNA-U repair in this organism.     

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity.     

Use of Mycobacterium smegmatis Deficient in ADP-Ribosyltransferase as Surrogate for Mycobacterium tuberculosis in Drug Testing and Mutation Analysis

Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.     

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

The reliable repair of pre-mutagenic U/G mismatches that originated from hydrolytic cytosine deamination is crucial for the maintenance of the correct genomic information. In most organisms, any uracil base in DNA is attacked by uracil DNA glycosylases (UDGs), but at least in Methanothermobacter thermautotrophicus ΔH, an alternative strategy has evolved. The exonuclease III homologue Mth212 from the thermophilic archaeon M. thermautotrophicus ΔH exhibits a DNA uridine endonuclease activity in addition to the apyrimidinic/apurinic site endonuclease and 3′ → 5′exonuclease functions. Mth212 alone compensates for the lack of a UDG in a single-step reaction thus substituting the two-step pathway that requires the consecutive action of UDG and apyrimidinic/apurinic site endonuclease.In order to gain deeper insight into the structural basis required for the specific uridine recognition by Mth212, we have characterized the enzyme by means of X-ray crystallography. Structures of Mth212 wild-type or mutant proteins either alone or in complex with DNA substrates and products have been determined to a resolution of up to 1.2 Å, suggesting key residues for the uridine endonuclease activity. The insertion of the side chain of Arg209 into the DNA helical base stack resembles interactions observed in human UDG and seems to be crucial for the uridine recognition. In addition, Ser171, Asn153, and Lys125 in the substrate binding pocket appear to have important functions in the discrimination of aberrant uridine against naturally occurring thymidine and cytosine residues in double-stranded DNA.     

Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4

Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.     

Compromised base excision repair pathway in Mycobacterium tuberculosis imparts superior adaptability in the host

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is a significant public health concern, exacerbated by the emergence of drug-resistant TB. To combat the host’s dynamic environment, Mtb encodes multiple DNA repair enzymes that play a critical role in maintaining genomic integrity. Mtb possesses a GC-rich genome, rendering it highly susceptible to cytosine deaminations, resulting in the occurrence of uracils in the DNA. UDGs encoded by ung and udgB initiate the repair; hence we investigated the biological impact of deleting UDGs in the adaptation of pathogen. We generated gene replacement mutants of uracil DNA glycosylases, individually (RvΔung, RvΔudgB) or together (RvΔdKO). The double KO mutant, RvΔdKO exhibited remarkably higher spontaneous mutation rate, in the presence of antibiotics. Interestingly, RvΔdKO showed higher survival rates in guinea pigs and accumulated large number of SNPs as revealed by whole-genome sequence analysis. Competition assays revealed the superior fitness of RvΔdKO over Rv, both in ex vivo and in vivo conditions. We propose that compromised DNA repair results in the accumulation of mutations, and a subset of these drives adaptation in the host. Importantly, this property allowed us to utilize RvΔdKO for the facile identification of drug targets.     

Complete Genome Sequence of Methanothermobacter marburgensis,a Methanoarchaeon Model Organism

The circular genome sequence of the chemolithoautotrophic euryarchaeon Methanothermobacter marburgensis, with 1,639,135 bp, was determined and compared with that of Methanothermobacter thermautotrophicus. The genomes of the two model methanogens differ substantially in protein coding sequences, in insertion sequence (IS)-like elements, and in clustered regularly interspaced short palindromic repeats (CRISPR) loci.Methanothermobacter marburgensis (DSM 2133) (formerly Methanobacterium thermoautotrophicum strain Marburg), a member of the Methanobacteriales (2), was isolated in 1978 from anaerobic sewage sludge in Marburg, Germany (5). The hydrogenotrophic methanogen grows even faster (2 h versus 3 h doubling time) and to higher cell concentrations (3 g versus 1.5 g dry mass per liter) than Methanothermobacter thermautotrophicus (DSM 1053) (formerly Methanobacterium thermoautotrophicum strain ΔH) (20) (for other differences, see references 3 and 19). Both methanogens were used in the last 35 years for the elucidation of the enzymes and coenzymes involved in CO₂ reduction to methane with H₂ (4, 16-18). The genome sequence of M. thermautotrophicus was reported in 1997 (15); that of M. marburgensis is announced here.The genome size of M. marburgensis is 1,639,135 bp (that of M. thermautotrophicus is 1,751,377 bp), the genome G+C content is 48.64% (49.54% for M. thermautotrophicus), and the part coding is 90.94% (91.02% for M. thermautotrophicus). Comparison of the sequences (13) revealed that the two genomes have 1,607 protein coding sequences (CDS) in common and 411 CDS not in common (145 CDS are found only in M. marburgensis and 266 CDS only in M. thermautotrophicus) and show a high degree of synteny. The CDS not in common could be traced back to gene splitting (15%), gene deletion (30%), gene duplication (30%), and lateral gene transfer (24%) events (percentages given are for M. marburgensis). Of the 1,607 CDS in common, approximately 40% show BLAST search expectation values of >10⁻¹⁰⁰ at the protein level, reflecting large differences in sequence divergence. Almost 470 CDS encode conserved hypothetical proteins.The genome of M. marburgensis harbors 15 insertion sequence (IS)-like elements, whereas there is no evidence for a classically organized IS-like element in M. thermautotrophicus. Consistently, a CDS for a transposase is found only in M. marburgensis.In the genome of M. marburgensis there is only one clustered regularly interspaced short palindromic repeat (CRISPR) locus with 36 repeats and only one CRISPR-associated (cas) gene (csa3), indicating that the organism is not protected from invasion by phage and plasmid DNA (7, 8, 10, 12). By comparison, in the genome of M. thermautotrophicus there are three CRISPR loci with 124, 4, and 47 repeats and 18 cas genes that encode proteins involved in adaptation and interference (http://genoweb1.irisa.fr/Serveur-GPO/outils/repeatsAnalysis/CRISPR/). The spacer sequences from locus 2 match DNA sequences found in phage ΨM1 of M. marburgensis (6, 11) and ΨM100 of M. wolfei (9), which supports the observation that M. thermautotrophicus is not lysed by those two phages. Unfortunately, there is no DNA sequence available for phage ΦF1, which is able to lyse M. thermautotrophicus (14), to compare it with the spacer sequences of the CRISPR regions. In the plasmid pM2001 (= pMTBMA4) (4,439-bp circular multicopy plasmid found only in M. marburgensis) (1, 19), no sequence identities for CRISPR spacer sequences of M. thermautotrophicus were found (14).Approximately 200 CDS were identified that are required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO₂ reduction to methane and in the coupling of this process with energy conservation. Some of the genes have been found only recently; others, such as those for coenzyme F₄₃₀ biosynthesis, still remain to be discovered.     

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101^Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101^Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.     

Mms22p protects Saccharomyces cerevisiae from DNA damage induced by topoisomerase II

The cleavage reaction of topoisomerase II, which creates double-stranded DNA breaks, plays a central role in both the cure and initiation of cancer. Therefore, it is important to understand the cellular processes that repair topoisomerase II-generated DNA damage. Using a genome-wide approach with Saccharomyces cerevisiae, we found that Δmre11, Δxrs2, Δrad50, Δrad51, Δrad52, Δrad54, Δrad55, Δrad57 and Δmms22 strains were hypersensitive to etoposide, a drug that specifically increases levels of topoisomerase II-mediated DNA breaks. These results confirm that the single-strand invasion pathway of homologous recombination is the major pathway that repairs topoisomerase II-induced DNA damage in yeast and also indicate an important role for Mms22p. Although Δmms22 strains are sensitive to several DNA-damaging agents, little is known about the function of Mms22p. Δmms22 cultures accumulate in G₂/M, and display an abnormal cell cycle response to topoisomerase II-mediated DNA damage. MMS22 appears to function outside of the single-strand invasion pathway, but levels of etoposide-induced homologous recombination in Δmms22 cells are lower than wild-type. MMS22 is epistatic with RTT101 and RTT107, genes that encode its protein binding partners. Finally, consistent with a role in DNA processes, Mms22p localizes to discrete nuclear foci, even in the absence of etoposide or its binding partners.     

A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair

Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.     

Role of DNA mismatch repair and double-strand break repair in genome stability and antifungal drug resistance in Candida albicans

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Δ/mre11Δ, rad50Δ/rad50Δ, and rad52Δ/rad52Δ mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Δ/msh2Δ, pms1Δ/pms1Δ, and yku80Δ/yku80Δ mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Δ/mre11Δ and rad50Δ/rad50Δ mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Δ/mre11Δ, rad50Δ/rad50Δ, or rad52Δ/rad52Δ leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Δ/msh2Δ and pms1Δ/pms1Δ (MMR mutants) and rad50Δ/rad50Δ (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.     

A Reversible Histone H3 Acetylation Cooperates with Mismatch Repair and Replicative Polymerases in Maintaining Genome Stability

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.     

Phosphatase Complex Pph3/Psy2 Is Involved in Regulation of Efficient Non-Homologous End-Joining Pathway in the Yeast Saccharomyces cerevisiae

One of the main mechanisms for double stranded DNA break (DSB) repair is through the non-homologous end-joining (NHEJ) pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.     

Energetic signatures of single base bulges: thermodynamic consequences and biological implications

DNA bulges are biologically consequential defects that can arise from template-primer misalignments during replication and pose challenges to the cellular DNA repair machinery. Calorimetric and spectroscopic characterizations of defect-containing duplexes reveal systematic patterns of sequence-context dependent bulge-induced destabilizations. These distinguishing energetic signatures are manifest in three coupled characteristics, namely: the magnitude of the bulge-induced duplex destabilization (ΔΔG_Bulge); the thermodynamic origins of ΔΔG_Bulge (i.e. enthalpic versus entropic); and, the cooperativity of the duplex melting transition (i.e. two-state versus non-two state). We find moderately destabilized duplexes undergo two-state dissociation and exhibit ΔΔG_Bulge values consistent with localized, nearest neighbor perturbations arising from unfavorable entropic contributions. Conversely, strongly destabilized duplexes melt in a non-two-state manner and exhibit ΔΔG_Bulge values consistent with perturbations exceeding nearest-neighbor expectations that are enthalpic in origin. Significantly, our data reveal an intriguing correlation in which the energetic impact of a single bulge base centered in one strand portends the impact of the corresponding complementary bulge base embedded in the opposite strand. We discuss potential correlations between these bulge-specific differential energetic profiles and their overall biological implications in terms of DNA recognition, repair and replication.