Glutamine tRNA Research Articles

Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells.

The Type VI secretion system (T6SS) is a bacterial nanomachine which delivers toxic effectors to kill competitors or subvert some of their key functions. Here we use transposon directed insertion-site sequencing (TraDIS) to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine tRNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis proteins and validate the use of a TraDIS-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.

Degradation of host translational machinery drives tRNA acquisition in viruses.

Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p= 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.

The identities of stop codon reassignments support ancestral tRNA stop codon decoding activity as a facilitator of gene duplication and evolution of novel function

Stop codon reassignments are widely distributed in prokaryotic, eukaryotic and organellar genomes, but are remarkably convergent in terms of the stop codons and amino acids reassigned. Strikingly, the identities of stop codon reassignments are closely matched to the properties of naturally occurring nonsense suppressor (NONS) tRNAs, suggesting that pre-existing nonsense suppression in an ancestral tRNA facilitated the occurrence of stop codon reassignments. Here this idea is expanded, by exploring the mechanism by which the gene duplication of tRNAs has occurred, leading to the reassignment of stop codons. Two types of stop codon reassignment are identified: those that necessitate a tRNA gene duplication, and those that do not because a single tRNA can recognize the reassigned stop codon and the canonical codon(s) for the cognate amino acid. Where tRNA gene duplication has occurred, this implies a multi-functional ancestral NONS tRNA, followed by adaptive mutation in the anticodon of one of the gene duplicates to become complementary to the stop codon, constituting a clear example of escape from adaptive conflict. The best exemplar is the UAA+UAG →gln reassignment, which has occurred 9 times independently in a diverse range of genomes, and appears to reflect the widespread occurrence of naturally occurring nonsense suppression of the UAA+UAG stop codons by glutamine tRNAs. Consideration of pre-existing tRNA functionality and the mechanism of gene duplication provide new insights into the process of stop codon reassignment.

Glutamine tRNA CUG ‐Dependent, Nitrogen‐Responsive Nuclear Gln3 and Gat1 Localization

A leucine-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis. However, Gln3 and Gat1, nitrogen-responsive transcription activators, don't respond to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down regulation of Gln3 occurs via a second mechanism. A major site of Gln3/Gat1 control occurs at their access to the nucleus. In excess nitrogen, Gln3/Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear when nitrogen or glutamine is limiting. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis by methionine sulfoximine (Msx) prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on Gln3/Gat1 regulation. Contrary to prediction, we show nuclear Gln3 localization - elicited by short- and long-term nitrogen starvation, growth in proline, during Msx or rapamycin treatment or in a ure2Δ - is abolished by alteration of glutamine tRNACUG. In contrast, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation, growth in proline medium or a ure2Δ, neither responds to Msx or long-term starvation nor requires tRNACUG for its response to rapamycin. Gln3/Gat1 requirements for the rare tRNACUG, cannot be replaced in growing cells by the predominant glutamine tRNACAA. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3/Gat1 localization that is highly sensitive to the function of the rare glutamine tRNACUG. Our data also demonstrate distinct mechanistic differences between the regulation of Gln3 and Gat1. Supported by NIH grant GM-35642.

Nitrogen starvation and TorC1 inhibition differentially affect nuclear localization of the Gln3 and Gat1 transcription factors through the rare glutamine tRNACUG in Saccharomyces cerevisiae.

A leucine, leucyl-tRNA synthetase-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors. This component is highly sensitive to the function of the rare glutamine tRNACUG, which cannot be replaced by the predominant glutamine tRNACAA. Our observations also demonstrate distinct mechanistic differences between the responses of Gln3 and Gat1 to rapamycin inhibition of TorC1 and nitrogen starvation.

Mutations in QARS, Encoding Glutaminyl-tRNA Synthetase, Cause Progressive Microcephaly, Cerebral-Cerebellar Atrophy, and Intractable Seizures

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.

Folate, vitamin B12 and homocystine status in the elderly in North-eastern Iran

Cystathionine β-synthase (CBS) deficiency is the most common cause of homocystinuria. More than 130 pathogenic mutations, mostly in the Caucasian populations, have been described. Recently, our group reported a mutation analysis of Japanese homocystinuric patients. In the present paper, we report an expression study of several mutant CBS enzymes in Escherichia coli, i.e., R121H, G148R, G151R, S217F, H232D, R266G, 1591delTTCG, and K441X. All of the mutants except K441X exhibited severely decreased activity, and the capability to form tetramers of most mutants was severely impaired. The K441X mutant, on the other hand, exhibited relatively high activity (63% of the wild type activity). This was probably due to two factors. First, the high abundance of the full-length CBS protein, a likely K441Q mutant, which was produced through suppression of the amber termination codon by glutamine tRNA in E. coli. And second, the presence of a C-terminally truncated protein, which was previously shown to be constitutively activated. Patient-derived lymphocytes, however, showed no detectable CBS subunits. As previously hypothesized, the increased aggregation of mutant CBS subunits might be a common pathogenic mechanism in CBS deficiency.

The conserved Wobble uridine tRNA thiolase Ctu1–Ctu2 is required to maintain genome integrity

Modified nucleosides close to the anticodon are important for the proper decoding of mRNA by the ribosome. Particularly, the uridine at the first anticodon position (U34) of glutamate, lysine, and glutamine tRNAs is universally thiolated (S(2)U34), which is proposed to be crucial for both restriction of wobble in the corresponding split codon box and efficient codon-anticodon interaction. Here we show that the highly conserved complex Ctu1-Ctu2 (cytosolic thiouridylase) is responsible for the 2-thiolation of cytosolic tRNAs in the nematode and fission yeast. In both species, inactivation of the complex leads to loss of thiolation on tRNAs and to a thermosensitive decrease of viability associated with marked ploidy abnormalities and aberrant development. Increased level of the corresponding tRNAs suppresses the fission yeast defects, and our data suggest that these defects could result from both misreading and frame shifting during translation. Thus, a translation defect due to unmodified tRNAs results in severe genome instability.

HIV-1 designed to use different tRNAGln isoacceptors prefers to select tRNAThr for replication

BackgroundPrevious studies have shown that infection with human immunodeficiency virus type 1 (HIV-1) causes acceleration of the synthesis of glutamine tRNA (tRNAGln) in infected cells. To investigate whether this might influence HIV-1 to utilize tRNAGln as a primer for initiation of reverse transcription, we have constructed HIV-1 proviral genomes in which the PBS and the A-loop region upstream of the PBS have been made complementary to either the anticodon region of tRNAGln,1 or tRNAGln,3 and 3' terminal 18 nucleotides of each isoacceptor of tRNAGln.ResultsViruses in which the PBS was altered to be complementary to tRNAGln,1 or tRNAGln,3 with or without the A-loop all exhibited a lower infectivity than the wild type virus. Viruses with only the PBS complementary to tRNAGln,1 or tRNAGln,3 reverted to wild type following culture in SupT1 cells. Surprisingly, viruses in which the PBS and A-loop were complementary to tRNAGln,1 did not grow in SupT1 cells, while viruses in which the PBS and A-loop were made complementary to tRNAGln,3 grew slowly in SupT1 cells. Analysis of the PBS of this virus revealed that it had reverted to select tRNAThr as the primer, which shares complementarity in 15 of 18 nucleotides with the PBS complementary to tRNAGln,3.ConclusionThe results of these studies support the concept that the HIV-1 has preferred tRNAs that can be selected as primers for replication.

Variation in evolutionary unstable regions of the chloroplast genome in plants obtained in anther culture of dihaploid wheat lines

In dihaploid wheats, two evolutionarily unstable regions of the chloroplast genome were examined. These regions include the following genes, changes in which could be associated with albinism in anther culture: rbcL, encoding the large Rubisco subunit; psaA, encoding p700 apoprotein Ia; petA, encoding cytochrome f; atpB and atpE, encoding respectively beta and epsilon subunits of the CF1 ATPase complex; trnE, encoding glutamine tRNA; and cemA, encoding a cell membrane protein. Using PCR, we have shown that atpB was the gene most often not detected in the lines examined. These results suggest that regeneration of albino plants is accompanied by a deletion of a chloroplast DNA region harboring this gene.

Expression study of mutant cystathionine β-synthase found in Japanese patients with homocystinuria

Cystathionine β-synthase (CBS) deficiency is the most common cause of homocystinuria. More than 130 pathogenic mutations, mostly in the Caucasian populations, have been described. Recently, our group reported a mutation analysis of Japanese homocystinuric patients. In the present paper, we report an expression study of several mutant CBS enzymes in Escherichia coli, i.e., R121H, G148R, G151R, S217F, H232D, R266G, 1591delTTCG, and K441X. All of the mutants except K441X exhibited severely decreased activity, and the capability to form tetramers of most mutants was severely impaired. The K441X mutant, on the other hand, exhibited relatively high activity (63% of the wild type activity). This was probably due to two factors. First, the high abundance of the full-length CBS protein, a likely K441Q mutant, which was produced through suppression of the amber termination codon by glutamine tRNA in E. coli. And second, the presence of a C-terminally truncated protein, which was previously shown to be constitutively activated. Patient-derived lymphocytes, however, showed no detectable CBS subunits. As previously hypothesized, the increased aggregation of mutant CBS subunits might be a common pathogenic mechanism in CBS deficiency.

Mitochondrial DNA sequences of the Afro-Arabian spiny-tailed lizards (genus Uromastyx; family Agamidae): phylogenetic analyses and evolution of gene arrangements

Approximately 1.7 kbp of mitochondrial DNA were sequenced from 29 individuals assignable to 11 Uromastyx species or subspecies and two other agamids. U. ocellata and U. ornata had an insertion between the glutamine and isoleucine tRNA genes, which could be folded into a stable stem-and-loop structure, and the insertion for U. ornata additionally retained a sequence similar to the glutamine tRNA gene. This corroborates the role of tandem duplication in reshaping mitochondrial gene arrangements, and supports the idea that the origin of light-strand replication could be relocated within mitochondrial genomes. Molecular phylogeny from different tree-building methods consistently placed African and Arabian taxa in mutually monophyletic groups, excluding U. hardwickii inhabiting India and Pakistan. Unlike previous studies based on morphology, U. macfadyeni did not cluster with morphologically similar Arabian taxa, suggesting convergent evolution to be responsible for the morphological similarities. Divergence times estimated among the Uromastyx taxa, together with geological and palaeontological evidence, suggest that the Uromastyx agamids originated from Central Asia during the Eocene and colonized Africa after its connection with Eurasia in the early Miocene. Their radiation may have been facilitated by repeated aridification of North Africa since the middle Miocene, and geological events such as the expansion of the Red Sea and the East African Rift Valley.

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells.

We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.

In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E. coli: a feasible part of UV-mutagenesis

We have estimated in vivo deamination rates for cytosines in cyclobutane pyrimidine dimers (CPD or Py ▪Py) in UV-irradiated E. coli deficient in uracil DNA glycosylase. The protocol consisted of UV-irradiation, holding in buffer to allow for deamination of cytosines in CPDs and photoreversal (PR) to establish uracils where cytosines in CPD deaminated. The deamination rate at T ▪C photoproducts targeting glutamine tRNA suppressor mutations was estimated from the increase in the mutation frequency after PR (MF PR) that developed as UV-irradiated cells were held before PR. Evidence suggested that an earlier study with this protocol under-estimated the deamination rate at sites producing the same mutations in an E. coli B/r strain. With a K12 strain, where the targeting apparently is principally by CPD and not (6–4) photoproducts, a larger rate of k = 0.0091 min −1 at 42 °C resulted. The dark assay for MF also increased significantly with time for deamination consistent with a model for efficient mutation by translesion synthesis at uracil-containing CPD. In addition, we used a strain constructed by Cupples and Miller in which β-galactosidase was inactive because -GGG- was at codon 461 and would revert to Lac + only when replaced by -GAG- or -GAA- for glutamate. C ▪C photoproducts at this target site in the opposite DNA strand could reveal effects of first and second deaminations in the same CPD. MF PR for Lac + mutations increased and then decreased as a function of deamination time (at six temperatures 36–48 °C). Fitting an approximate model equation that distinguished two different deamination rates to these data suggested a first deamination producing Lac + at a rate about eight-fold less than a second deamination restoring the Lac − phenotype. We conclude that deamination, changing a cytosine-containing CPD to a uracil-containing CPD, could be an integral part of UV-induced C-to-T mutations.

Mitochondrial genetic code in cestodes

The flatworm mitochondrial genetic code, which has been used for all species of the Platyhelminthes, is mainly characterized by AUA codon for isoleucine, AAA codon for asparagine and UAA codon for tyrosine. In eight species of cestodes ( Echinococcus multilocularis, Echinococcus granulosus, Taenia solium, Taenia saginata, Taenia hydatigena, Taenia crassiceps, Hymenolepis nana and Mesocestoides corti), the cytochrome c oxidase subunit I (COI) genes were partially sequenced to verify this genetic code. Comparison of the COI-encoding nucleotide sequences with those of human, sea urchin, fruit fly, nematode and yeast indicated that the assignments of AUA and AAA codons are adequate for cestodes. In addition, the nucleotide sequences of ATPase subunit 6 (ATP6) gene and its flanking region were compared to examine initiation and stop codons. In the related species of T. solium and T. saginata, the deduced amino acid sequences of ATP6 were homogeneous; however, the conversion of initiation codon AUG into GUG was observed in T. saginata. We also found the similar conversion in T. crassiceps. The C-terminal sequences of putative ATP6 proteins were highly conserved among the eight species and the stop codon UAG was altered to UAA in all Taenia species. The features of the gene-junctional region between NADH dehydrogenase subunit 4 (ND4) and glutamine tRNA (tRNA Gln) genes also supported that UAA serves as a stop codon. Based on these results, we propose that the flatworm mitochondrial code should be modified for cestodes, particularly, in an initiating methionine codon (GUG) and a terminating codon (UAA).

An engineered class I transfer RNA with a class II tertiary fold.

Structure-based engineering of the tertiary fold of Escherichia coli tRNA(Gln)2 has enabled conversion of this transfer RNA to a class II structure while retaining recognition properties of a class I glutamine tRNA. The new tRNA possesses the 20-nt variable stem-loop of Thermus thermophilus tRNA(Ser). Enlargement of the D-loop appears essential to maintaining a stable tertiary structure in this species, while rearrangement of a base triple in the augmented D-stem is critical for efficient glutaminylation. These data provide new insight into structural determinants distinguishing the class I and class II tRNA folds, and demonstrate a marked sensitivity of glutaminyl-tRNA synthetase to alteration of tRNA tertiary structure.

Transcriptional mutagenesis and DNA strand asymmetrical mutations expressed in Escherichia coli under restrictive metabolic conditions

Mutagenesis by C-to-U events specifically in either DNA strand assayed with ung uvrA defective Escherichia coli on a metabolically restrictive medium produces more glutamine tRNA suppressor mutations from U occurring in the non-transcribed DNA strand than from U in the transcribed (template) DNA strand. This bias is the reverse of what might be expected from transcriptional mutagenesis (mutation expression utilizing mutated RNA transcribed from damaged template strand DNA). The results and related ideas are discussed.

Major Identity Element of Glutamine tRNAs from Bacillus subtilis and Escherichia coli in the Reaction with B. subtilis Glutamyl-tRNA Synthetase

Major Identity Element of Glutamine tRNAs from Bacillus subtilis and Escherichia coli in the Reaction with B. subtilis Glutamyl-tRNA Synthetase

A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation.

Dimorphic growth of the budding yeast Saccharomyces cerevisiae is regulated by the quality of the nitrogen supply. On a preferred nitrogen source diploid cells grow as ellipsoidal cells by using a bipolar pattern of budding, whereas on a poor nitrogen source a unipolar pattern of budding is adopted, resulting in extended pseudohyphal chains of filamentous cells. Here we report that the quality of the nitrogen source is signaled by the glutamine tRNA isoform with a 5'-CUG anticodon (tRNACUG). Mutations that alter this tRNA impair assessment of the nitrogen supply without measurably affecting protein synthesis, so that mutant cells display pseudohyphal growth even on a preferred nitrogen source. The nitrogen status for other nitrogen-responsive processes such as catabolic gene expression and sporulation also is signaled by this tRNA: mutant cells inappropriately induce the nitrogen-repressed gene CAR1 and undergo precocious sporulation in nitrogen-rich media. Therefore, in addition to its role in mRNA translation, this tRNA also transduces nitrogen signals that regulate development.

Photoreversal of UV-potentiated glutamine tRNA suppressor mutations in excision proficient Escherichia coli

UV-irradiated excision proficient Escherichia coli were exposed to light for photoenzymatic reversal (PR) of cyclobutane pyrimidine dimers (CPD) and assayed for reversion mutation (glutamine tRNA suppressor mutations) on semi-enriched medium or on the same medium containing acriflavine to inhibit excision repair. The initial mutation frequency without PR was relatively greater when assayed with acriflavine, and this difference increased as larger UV fluences were used. The PR kinetics were first order and about the same or slightly faster when cells were assayed with acriflavine (after 15, 30 or 45 J/m2, respectively). The results indicated mutation targeting by CPD in excision proficient cells. These results and conclusion contrast sharply with the original study of this type done several years ago. PR kinetics were considerably slower with assays containing acriflavine, sustaining the idea that PR causes repair of non-dimer targeting lesions by enhancing excision repair. To explain this contrast we devised a fluence-decrement rate for estimating the effectiveness of PR and measured PR-dependent excision repair (PER) as the difference in the fluence-decrement rate with excision proficient and deficient cells. PER was more evident when cells were prepared as in the original study but was still an insufficient factor. More importantly, the original study included a component of indirect photoreactivation or photoprotection (using unfiltered PR light) which accentuated the role of excision repair. Taking these factors into account, the original data also are consistent with the model that glutamine tRNA suppressor mutations produced by UV-mutagenesis in excision proficient E. coli result from targeting by CPD just as in excision defective cells. Thus, with regards to a common UV mutation assay, there does not appear to be two types of targeting lesion depending on excision proficiency.