High-Level Production of Recombinant Snowdrop Lectin in Sugarcane and Energy Cane.

Sugarcane and energy cane (Saccharum spp. hybrids) are ideal for plant-based production of recombinant proteins because their high resource-use efficiency, rapid growth and efficient photosynthesis enable extensive biomass production and protein accumulation at a cost-effective scale. Here, we aimed to develop these species as efficient platforms to produce recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA), a monocot-bulb mannose-specific lectin with potent antiviral, antifungal and antitumor activities. Initially, GNA levels of 0.04% and 0.3% total soluble protein (TSP) (0.3 and 3.8 mg kg-1 tissue) were recovered from the culms and leaves, respectively, of sugarcane lines expressing recombinant GNA under the control of the constitutive maize ubiquitin 1 (Ubi) promoter. Co-expression of recombinant GNA from stacked multiple promoters (pUbi and culm-regulated promoters from sugarcane dirigent5-1 and Sugarcane bacilliform virus) on separate expression vectors increased GNA yields up to 42.3-fold (1.8% TSP or 12.7 mg kg-1 tissue) and 7.7-fold (2.3% TSP or 29.3 mg kg-1 tissue) in sugarcane and energy cane lines, respectively. Moreover, inducing promoter activity in the leaves of GNA transgenic lines with stress-regulated hormones increased GNA accumulation to 2.7% TSP (37.2 mg kg-1 tissue). Purification by mannose-agarose affinity chromatography yielded a functional sugarcane recombinant GNA with binding substrate specificity similar to that of native snowdrop-bulb GNA, as shown by enzyme-linked lectin and mannose-binding inhibition assays. The size and molecular weight of recombinant GNA were identical to those of native GNA, as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. This work demonstrates the feasibility of producing recombinant GNA at high levels in Saccharum species, with the long-term goal of using it as a broad-spectrum antiviral carrier molecule for hemopurifiers and in related therapeutic applications.

Plant-made E2 glycoprotein single-dose vaccine protects pigs against classical swine fever.

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at-risk animals, often at very high cost. Current CSFV-modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first-generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide-spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium-mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil-in-water emulsion adjuvants. We report the manufacturing of adjuvanted, plant-made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single-dose vaccination, which was accompanied by strong virus neutralization antibody responses.

Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies.

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or ß-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics' glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.

Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals.

Rapid, large-scale manufacture of medical countermeasures can be uniquely met by the plant-made-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plant-based manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever.

CSB interacts with SNM1A and promotes DNA interstrand crosslink processing.

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation.

Replication of nuclear DNA occurs in the context of chromatin and is influenced by histone modifications. In the ciliate Tetrahymena thermophila, we identified TXR1, encoding a histone methyltransferase. TXR1 deletion resulted in severe DNA replication stress, manifested by the accumulation of ssDNA, production of aberrant replication intermediates, and activation of robust DNA damage responses. Paired-end Illumina sequencing of ssDNA revealed intergenic regions, including replication origins, as hot spots for replication stress in ΔTXR1 cells. ΔTXR1 cells showed a deficiency in histone H3 Lys 27 monomethylation (H3K27me1), while ΔEZL2 cells, deleting a Drosophila E(z) homolog, were deficient in H3K27 di- and trimethylation, with no detectable replication stress. A point mutation in histone H3 at Lys 27 (H3 K27Q) mirrored the phenotype of ΔTXR1, corroborating H3K27me1 as a key player in DNA replication. Additionally, we demonstrated interactions between TXR1 and proliferating cell nuclear antigen (PCNA). These findings support a conserved pathway through which H3K27me1 facilitates replication elongation.

Human Cockayne syndrome B protein reciprocally communicates with mitochondrial proteins and promotes transcriptional elongation.

Cockayne syndrome (CS) is a rare human disorder characterized by pathologies of premature aging, neurological abnormalities, sensorineural hearing loss and cachectic dwarfism. With recent data identifying CS proteins as physical components of mitochondria, we sought to identify protein partners and roles for Cockayne syndrome group B (CSB) protein in this organelle. CSB was found to physically interact with and modulate the DNA-binding activity of the major mitochondrial nucleoid, DNA replication and transcription protein TFAM. Components of the mitochondrial transcription apparatus (mitochondrial RNA polymerase, transcription factor 2B and TFAM) all functionally interacted with CSB and stimulated its double-stranded DNA-dependent adenosine triphosphatase activity. Moreover, we found that patient-derived CSB-deficient cells exhibited a defect in efficient mitochondrial transcript production and that CSB specifically promoted elongation by the mitochondrial RNA polymerase in vitro. These observations provide strong evidence for the importance of CSB in maintaining mitochondrial function and argue that the pathologies associated with CS are in part, a direct result of the roles that CSB plays in mitochondria.

Pathways for repairing and tolerating the spectrum of oxidative DNA lesions.

Reactive oxygen species (ROS) arise from both endogenous and exogenous sources. These reactive molecules possess the ability to damage both the DNA nucleobases and the sugar phosphate backbone, leading to a wide spectrum of lesions, including non-bulky (8-oxoguanine and formamidopyrimidine) and bulky (cyclopurine and etheno adducts) base modifications, abasic sites, non-conventional single-strand breaks, protein-DNA adducts, and intra/interstrand DNA crosslinks. Unrepaired oxidative DNA damage can result in bypass mutagenesis during genome copying or gene expression, or blockage of the essential cellular processes of DNA replication or transcription. Such outcomes underlie numerous pathologies, including, but not limited to, carcinogenesis and neurodegeneration, as well as the aging process. Cells have adapted and evolved defense systems against the deleterious effects of ROS, and specifically devote a number of cellular DNA repair and tolerance pathways to combat oxidative DNA damage. Defects in these protective pathways trigger hereditary human diseases that exhibit increased cancer incidence, developmental defects, neurological abnormalities, and/or premature aging. We review herein classic and atypical oxidative DNA lesions, outcomes of encountering these damages during DNA replication and transcription, and the consequences of losing the ability to repair the different forms of oxidative DNA damage. We particularly focus on the hereditary human diseases Xeroderma Pigmentosum, Cockayne Syndrome and Fanconi Anemia, which may involve defects in the efficient repair of oxidative modifications to chromosomal DNA.

Characterization of the endoribonuclease active site of human apurinic/apyrimidinic endonuclease 1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg(2+), while ssDNA AP site cleavage required Mg(2+) (optimally at 0.5-2.0 mM). We also found that a 2'-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3'-PO(4)(2-) group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.

Variation in base excision repair capacity.

The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence variability in DNA repair genes and several studies have associated BER gene polymorphisms with disease risk, including cancer of several sites. The intent of this review is to describe the range of BER capacity among individuals and the functional consequences of BER genetic variants. We also discuss studies that associate BER deficiency with disease risk and the current state of BER capacity measurement assays.

Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population.

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLbeta, PARP-1, LIG3alpha, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLbeta, PARP-1, LIG3alpha and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants-excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression-exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLbeta-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks.

Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane.

Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases. Most patients belong to complementation group B, and the CS group B (CSB) protein plays a role in genomic maintenance and transcriptome regulation. By immunocytochemistry, mitochondrial fractionation, and Western blotting, we demonstrate that CSB localizes to mitochondria in different types of cells, with increased mitochondrial distribution following menadione-induced oxidative stress. Moreover, our results suggest that CSB plays a significant role in mitochondrial base excision repair (BER) regulation. In particular, we find reduced 8-oxo-guanine, uracil, and 5-hydroxy-uracil BER incision activities in CSB-deficient cells compared to wild-type cells. This deficiency correlates with deficient association of the BER activities with the mitochondrial inner membrane, suggesting that CSB may participate in the anchoring of the DNA repair complex. Increased mutation frequency in mtDNA of CSB-deficient cells demonstrates functional significance of the presence of CSB in the mitochondria. The results in total suggest that CSB plays a direct role in mitochondrial BER by helping recruit, stabilize, and/or retain BER proteins in repair complexes associated with the inner mitochondrial membrane, perhaps providing a novel basis for understanding the complex phenotype of this debilitating disorder.

Stoichiometry of base excision repair proteins correlates with increased somatic CAG instability in striatum over cerebellum in Huntington's disease transgenic mice.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5'-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 5'-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP-BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLbeta was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLbeta strand displacement activity during LP-BER promotes the formation of stable 5'-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical factor underlying the tissue selectivity of somatic CAG expansion.

Nucleic acid binding activity of human Cockayne syndrome B protein and identification of Ca(2+) as a novel metal cofactor.

The Cockayne syndrome group B protein (CSB) is a member of the SWI/SNF2 subgroup of Superfamily 2 ATPases/nucleic acid translocases/helicases and is defective in the autosomal recessive segmental progeroid disorder Cockayne syndrome. This study examines the ATP-dependent and the ATP-independent biochemical functions of human CSB. We show that Ca(2+) is a novel metal cofactor of CSB for ATP hydrolysis, mainly through the enhancement of k(cat), and that a variety of biologically relevant model nucleic acid substrates can function to activate CSB ATPase activity with either Mg(2+) or Ca(2+) present. However, CSB lacked detectable ATP-dependent helicase and single- or double-stranded nucleic acid translocase activities in the presence of either divalent metal. CSB was found to support ATP-independent complementary strand annealing of DNA/DNA, DNA/RNA, and RNA/RNA duplexes, with Ca(2+) again promoting optimal activity. CSB formed a stable protein:DNA complex with a 34mer double-stranded DNA in electrophoretic mobility-shift assays, independent of divalent metal or nucleotide (e.g. ATP). Moreover, CSB was able to form a stable complex with a range of nucleic acid substrates, including bubble and "pseudo-triplex" double-stranded DNAs that resemble replication and transcription intermediates, as well as forked duplexes of DNA/DNA, DNA/RNA, and RNA/RNA composition, the latter two of which do not promote CSB ATPase activity. Association of CSB with DNA, independent of ATP binding or hydrolysis, was seemingly sufficient to displace or rearrange a stable pre-bound protein:DNA complex, a property potentially important for its roles in transcription and DNA repair.

Multiple replication origins of Halobacterium sp. strain NRC-1: properties of the conserved orc7-dependent oriC1.

The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.

Characterization of abasic endonuclease activity of human Ape1 on alternative substrates, as well as effects of ATP and sequence context on AP site incision.

Human Ape1 is a multifunctional protein with a major role in initiating repair of apurinic/apyrimidinic (AP) sites in DNA by catalyzing hydrolytic incision of the phosphodiester backbone immediately adjacent to the damage. Besides in double-stranded DNA, Ape1 has been shown to cleave at AP sites in single-stranded regions of a number of biologically relevant DNA conformations and in structured single-stranded DNA. Extension of these studies has revealed a more expansive repertoire of model substrates on which Ape1 exerts AP endonuclease activity. In particular, Ape1 possesses the ability to cleave at AP sites located in (i) the DNA strand of a DNA/RNA hybrid, (ii) "pseudo-triplex" bubble substrates designed to mimic stalled replication or transcription intermediates, and (iii) configurations that emulate R-loop structures that arise during class switch recombination. Moreover, Ape1 was found to cleave AP-site-containing single-stranded RNA, suggesting a novel "cleansing" function that may contribute to the elimination of detrimental cellular AP-RNA molecules. Finally, sequence context immediately surrounding an abasic site in duplex DNA was found to have a less than threefold effect on the incision efficiency of Ape1, and ATP was found to exert complex effects on the endonuclease capacity of Ape1 on double-stranded substrates. The results suggest that in addition to abasic sites in conventional duplex genomic DNA, Ape1 has the ability to incise at AP sites in DNA conformations formed during DNA replication, transcription, and class switch recombination, and that Ape1 can endonucleolytically destroy damaged RNA.

Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1.

BACKGROUND: Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. RESULTS: Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence). The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. CONCLUSION: The results showed that ten out of nineteen eukaryotic-type DNA replication genes are essential for Halobacterium sp. NRC-1, consistent with their requirement for DNA replication. The essential genes code for two of ten Orc/Cdc6 proteins, two out of three DNA polymerases, the MCM helicase, two DNA primase subunits, the DNA polymerase sliding clamp, and the flap endonuclease.

The uvrA, uvrB and uvrC genes are required for repair of ultraviolet light induced DNA photoproducts in Halobacterium sp. NRC-1.

BACKGROUND: Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants. RESULTS: Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6-4 photoproducts from their DNA after irradiation with 150 J/m2 of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry. CONCLUSION: Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6-4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.