Disruption of Spermatogenesis and Infertility in Ataxia with Oculomotor Apraxia Type 2 (AOA2).

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia characterized by onset between 10 and 20 years of age and a range of neurological features that include progressive cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia in a majority of patients, and elevated serum alpha-fetoprotein (AFP). AOA2 is caused by mutation of the SETX gene which encodes senataxin, a DNA/RNA helicase involved in transcription regulation, RNA processing, and DNA maintenance. Disruption of senataxin in rodents led to defective spermatogenesis and sterility in males uncovering a key role for senataxin in male germ cell survival. Here, we report the first clinical and cellular evidence of impaired spermatogenesis in AOA2 patients. We assessed sperm production in three AOA2 patients and testicular pathology in one patient and compared the findings to those of Setx-knockout mice. Sperm production was impaired in all patients assessed (3/3, 100%). Analyses of testicular biopsies from an AOA2 patient recapitulate features of the histology seen in Setx-knockout mice, strongly suggesting an underlying mechanism centering on DNA-damage-mediated germ cell apoptosis. These findings support a role for senataxin in human reproductive function and highlight a novel clinical feature of AOA2 that extends the extra-neurological roles of senataxin. This raises an important reproductive counseling issue for clinicians, and fertility specialists should be aware of SETX mutations as a possible diagnosis in young male patients presenting with oligospermia or azoospermia since infertility may presage the later onset of neurological manifestations in some individuals.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.

A new model to study neurodegeneration in ataxia oculomotor apraxia type 2.

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia. Recent evidence suggests that the protein defective in this syndrome, senataxin (SETX), functions in RNA processing to protect the integrity of the genome. To date, only patient-derived lymphoblastoid cells, fibroblasts and SETX knockdown cells were available to investigate AOA2. Recent disruption of the Setx gene in mice did not lead to neurobehavioral defects or neurodegeneration, making it difficult to study the etiology of AOA2. To develop a more relevant neuronal model to study neurodegeneration in AOA2, we derived neural progenitors from a patient with AOA2 and a control by induced pluripotent stem cell (iPSC) reprogramming of fibroblasts. AOA2 iPSC and neural progenitors exhibit increased levels of oxidative damage, DNA double-strand breaks, increased DNA damage-induced cell death and R-loop accumulation. Genome-wide expression and weighted gene co-expression network analysis in these neural progenitors identified both previously reported and novel affected genes and cellular pathways associated with senataxin dysfunction and the pathophysiology of AOA2, providing further insight into the role of senataxin in regulating gene expression on a genome-wide scale. These data show that iPSCs can be generated from patients with the autosomal recessive ataxia, AOA2, differentiated into neurons, and that both cell types recapitulate the AOA2 cellular phenotype. This represents a novel and appropriate model system to investigate neurodegeneration in this syndrome.

Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis.

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.

Senataxin controls meiotic silencing through ATR activation and chromatin remodeling.

Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA-DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx (-/-) pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration.

Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2.

Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing. To determine whether AOA2 and ALS4 mutations differentially affect gene expression, we overexpressed disease-specific SETX mutations in senataxin-haploinsufficient fibroblasts and observed changes in distinct sets of genes. This implicates mutation-specific alterations of senataxin function in disease pathogenesis and provides a novel example of allelic neurogenetic disorders with differing gene expression profiles. Weighted gene co-expression network analysis (WGCNA) demonstrated these senataxin-associated genes to be involved in both mutation-specific and shared functional gene networks. To assess this in vivo, we performed gene expression analysis on peripheral blood from members of 12 different AOA2 families and identified an AOA2-specific transcriptional signature. WGCNA identified two gene modules highly enriched for this transcriptional signature in the peripheral blood of all AOA2 patients studied. These modules were disease-specific and preserved in patient fibroblasts and in the cerebellum of Setx knockout mice demonstrating conservation across species and cell types, including neurons. These results identify novel genes and cellular pathways related to senataxin function in normal and disease states, and implicate alterations in gene expression as underlying the phenotypic differences between AOA2 and ALS4.

R-loops in proliferating cells but not in the brain: implications for AOA2 and other autosomal recessive ataxias.

Disruption of the Setx gene, defective in ataxia oculomotor apraxia type 2 (AOA2) leads to the accumulation of DNA/RNA hybrids (R-loops), failure of meiotic recombination and infertility in mice. We report here the presence of R-loops in the testes from other autosomal recessive ataxia mouse models, which correlate with fertility in these disorders. R-loops were coincident in cells showing high basal levels of DNA double strand breaks and in those cells undergoing apoptosis. Depletion of Setx led to high basal levels of R-loops and these were enhanced further by DNA damage both in vitro and in vivo in tissues with proliferating cells. There was no evidence for accumulation of R-loops in the brains of mice where Setx, Atm, Tdp1 or Aptx genes were disrupted. These data provide further evidence for genome destabilization as a consequence of disrupted transcription in the presence of DNA double strand breaks arising during DNA replication or recombination. They also suggest that R-loop accumulation does not contribute to the neurodegenerative phenotype in these autosomal recessive ataxias.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Senataxin protects the genome: Implications for neurodegeneration and other abnormalities.

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive disorder characterized by cerebellar atrophy, peripheral neuropathy, loss of Purkinje cells and elevated α-fetoprotein. AOA2 is caused by mutations in the SETX gene that codes for the high molecular weight protein senataxin. Mutations in this gene also cause dominant neurodegenerative disorders. Similar to that observed for other autosomal recessive ataxias, this protein protects the integrity of the genome against oxidative and other forms of DNA damage to reduce the risk of neurodegeneration. Senataxin functions in transcription termination and RNA splicing and it has been shown to resolve RNA/DNA hybrids (R-loops) that arise at transcription pause sites or when transcription is blocked. Recent data suggest that this protein functions at the interface between transcription and DNA replication to minimise the risk of collision and maintain genome stability. Our recent data using SETX gene-disrupted mice revealed that male mice were defective in spermatogenesis and were infertile. DNA double strand-breaks persisted throughout meiosis and crossing-over failed in SETX mutant mice. These changes can be explained by the accumulation of R-loops, which interfere with Holiday junctions and crossing-over. We also showed that senataxin was localized to the XY body in pachytene cells and was involved in transcriptional silencing of these chromosomes. While the defect in meiotic recombination was striking in these animals, there was no evidence of neurodegeneration as observed in AOA2 patients. We discuss here potentially different roles for senataxin in proliferating and post-mitotic cells.

ATM protein-dependent phosphorylation of Rad50 protein regulates DNA repair and cell cycle control.

The Mre11/Rad50/NBN complex plays a central role in coordinating the cellular response to DNA double-strand breaks. The importance of Rad50 in that response is evident from the recent description of a patient with Rad50 deficiency characterized by chromosomal instability and defective ATM-dependent signaling. We report here that ATM (defective in ataxia-telangiectasia) phosphorylates Rad50 at a single site (Ser-635) that plays an important adaptor role in signaling for cell cycle control and DNA repair. Although a Rad50 phosphosite-specific mutant (S635G) supported normal activation of ATM in Rad50-deficient cells, it was defective in correcting DNA damage-induced signaling through the ATM-dependent substrate SMC1. This mutant also failed to correct radiosensitivity, DNA double-strand break repair, and an S-phase checkpoint defect in Rad50-deficient cells. This was not due to disruption of the Mre11/Rad50/NBN complex revealing for the first time that phosphorylation of Rad50 plays a key regulatory role as an adaptor for specific ATM-dependent downstream signaling through SMC1 for DNA repair and cell cycle checkpoint control in the maintenance of genome integrity.

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response.

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.

Aprataxin, poly-ADP ribose polymerase 1 (PARP-1) and apurinic endonuclease 1 (APE1) function together to protect the genome against oxidative damage.

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1 (AOA1), is a DNA repair protein that processes the product of abortive ligations, 5' adenylated DNA. In addition to its interaction with the single-strand break repair protein XRCC1, aprataxin also interacts with poly-ADP ribose polymerase 1 (PARP-1), a key player in the detection of DNA single-strand breaks. Here, we reveal reduced expression of PARP-1, apurinic endonuclease 1 (APE1) and OGG1 in AOA1 cells and demonstrate a requirement for PARP-1 in the recruitment of aprataxin to sites of DNA breaks. While inhibition of PARP activity did not affect aprataxin activity in vitro, it retarded its recruitment to sites of DNA damage in vivo. We also demonstrate the presence of elevated levels of oxidative DNA damage in AOA1 cells coupled with reduced base excision and gap filling repair efficiencies indicative of a synergy between aprataxin, PARP-1, APE-1 and OGG1 in the DNA damage response. These data support both direct and indirect modulating functions for aprataxin on base excision repair.

Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation.

Ataxia oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin, a putative DNA/RNA helicase which shares high homology to the yeast Sen1p protein and has been shown to play a role in the response to oxidative stress. To investigate further the function of senataxin, we identified novel senataxin-interacting proteins, the majority of which are involved in transcription and RNA processing, including RNA polymerase II. Binding of RNA polymerase II to candidate genes was significantly reduced in senataxin deficient cells and this was accompanied by decreased transcription of these genes, suggesting a role for senataxin in the regulation/modulation of transcription. RNA polymerase II-dependent transcription termination was defective in cells depleted of senataxin in keeping with the observed interaction of senataxin with poly(A) binding proteins 1 and 2. Splicing efficiency of specific mRNAs and alternate splice-site selection of both endogenous genes and artificial minigenes were altered in senataxin depleted cells. These data suggest that senataxin, similar to its yeast homolog Sen1p, plays a role in coordinating transcriptional events, in addition to its role in DNA repair.

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage.

A defective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.

Nucleolar localization of aprataxin is dependent on interaction with nucleolin and on active ribosomal DNA transcription.

The APTX gene, mutated in patients with the neurological disorder ataxia with oculomotor apraxia type 1 (AOA1), encodes a novel protein aprataxin. We describe here, the interaction and interdependence between aprataxin and several nucleolar proteins, including nucleolin, nucleophosmin and upstream binding factor-1 (UBF-1), involved in ribosomal RNA (rRNA) synthesis and cellular stress signalling. Interaction between aprataxin and nucleolin occurred through their respective N-terminal regions. In AOA1 cells lacking aprataxin, the stability of nucleolin was significantly reduced. On the other hand, down-regulation of nucleolin by RNA interference did not affect aprataxin protein levels but abolished its nucleolar localization suggesting that the interaction with nucleolin is involved in its nucleolar targeting. GFP-aprataxin fusion protein co-localized with nucleolin, nucleophosmin and UBF-1 in nucleoli and inhibition of ribosomal DNA transcription altered the distribution of aprataxin in the nucleolus, suggesting that the nature of the nucleolar localization of aprataxin is also dependent on ongoing rRNA synthesis. In vivo rRNA synthesis analysis showed only a minor decrease in AOA1 cells when compared with controls cells. These results demonstrate a cross-dependence between aprataxin and nucleolin in the nucleolus and while aprataxin does not appear to be directly involved in rRNA synthesis its nucleolar localization is dependent on this synthesis.

Defective p53 response and apoptosis associated with an ataxia-telangiectasia-like phenotype.

Ataxia-telangiectasia mutated (ATM), the protein defective in ataxia-telangiectasia, plays a central role in DNA damage response and signaling to cell cycle checkpoints. We describe here a cell line from a patient with an ataxia-telangiectasia-like clinical phenotype defective in the p53 response to radiation but with normal ATM activation and efficient downstream phosphorylation of other ATM substrates. No mutations were detected in ATM cDNA. A normal level of interaction between p53 and peptidyl-prolyl-isomerase Pin1 suggests that posttranslational modification was intact in these cells but operating at reduced level. Defective p53 stabilization was accompanied by defective induction of p53 effector genes and failure to induce apoptosis in response to DNA-damaging agents. Continued association between p53 and murine double minute-2 (Mdm2) occurred in irradiated ATL2ABR cells in response to DNA damage, and incubation with Mdm2 antagonists, nutlins, increased the stabilization of p53 and its transcriptional activity but failed to induce apoptosis. These results suggest that ATM-dependent stabilization of p53 and induction of apoptosis by radiation involve an additional factor(s) that is defective in ATL2ABR cells.

Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase.

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.

Aprataxin, a novel protein that protects against genotoxic stress.

Ataxia-oculomotor apraxia (AOA1) is a neurological disorder with symptoms that overlap those of ataxia-telangiectasia, a syndrome characterized by abnormal responses to double-strand DNA breaks and genome instability. The gene mutated in AOA1, APTX, is predicted to code for a protein called aprataxin that contains domains of homology with proteins involved in DNA damage signalling and repair. We demonstrate that aprataxin is a nuclear protein, present in both the nucleoplasm and the nucleolus. Mutations in the APTX gene destabilize the aprataxin protein, and fusion constructs of enhanced green fluorescent protein and aprataxin, representing deletions of putative functional domains, generate highly unstable products. Cells from AOA1 patients are characterized by enhanced sensitivity to agents that cause single-strand breaks in DNA but there is no evidence for a gross defect in single-strand break repair. Sensitivity to hydrogen peroxide and the resulting genome instability are corrected by transfection with full-length aprataxin cDNA. We also demonstrate that aprataxin interacts with the repair proteins XRCC1, PARP-1 and p53 and that it co-localizes with XRCC1 along charged particle tracks on chromatin. These results demonstrate that aprataxin influences the cellular response to genotoxic stress very likely by its capacity to interact with a number of proteins involved in DNA repair.

Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis.

In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5'-DNA end. DnaE is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.

Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition.

Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD' and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD' and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD' and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.