Evidence that nucleotide excision repair is attenuated in bax-deficient mammalian cells following ultraviolet irradiation

Nonmelanoma skin cancer (NMSC) is the most frequently diagnosed form of cancer in United States. We have previously described the tumor suppressor role of bax protein in skin carcinogenesis as well as the ability of bax to modulate the apoptotic response of keratinocytes following ultraviolet irradiation. Moreover, we have demonstrated an increase in tumor incidence in bax-null mice compared to control littermates in an in vivo chemical carcinogenesis experiment. In this study, we examined the contribution of bax protein in repair of UVR-mediated DNA lesions. The level of cyclobutane pyrimidine dimers remaining was twofold greater in UVR-treated primary keratinocytes from bax-deficient mice than that of control cells at 48 h (P < 0.03). Similar results were obtained using embryonic fibroblasts and also with a bax-deficient prostate cancer cell line. However, the repair rate of 6-4 pyrimidine pyrimidone photoproducts was unaffected by the absence of bax protein. These findings suggest that bax may have a dual function in its role as tumor suppressor in NMSC. Bax may directly or indirectly facilitate either DNA repair or cell death. Either function would be anticipated to enhance genomic integrity following a genotoxic event through repair or deletion of the damaged cell.     

Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue.     

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair.

Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S. cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.     

Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.     

DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6–4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.     

Variability in nucleotide excision repair and cancer risk: a review

Cancer initiation is classically associated with the induction of mutations on specific oncogenes or tumor suppressor genes, due to the presence of unrepaired DNA lesions produced by endogenous or exogenous genotoxic agents. Among several DNA repair pathways, the nucleotide excision repair (NER) is the most important and versatile one in removing the bulky adducts induced by physical and chemical carcinogens. Xeroderma pigmentosum (XP), characterized by a deficiency in NER and an over 1000-fold increased risk of skin cancer, represents a paradigm to understand the role of unrepaired lesion in the development of cancer. We reviewed here several NER assays used in epidemiological studies investigating the association between DNA repair efficiency and cancer risk. Reduced DNA repair could contribute to the development of cutaneous basal cell carcinoma (BCC), although discordant results have been reported. More consistent findings were observed between cellular sensitivity towards genotoxic agents and smoking-related cancers.     

Cytosolic iron‐sulphur protein assembly is functionally conserved and essential in procyclic and bloodstream Trypanosoma brucei

Cytosolic and nuclear iron‐sulphur (Fe/S) proteins include essential components involved in protein translation, DNA synthesis and DNA repair. In yeast and human cells, assembly of their Fe/S cofactor is accomplished by the CIA (cytosolic iron‐sulphur protein assembly) machinery comprised of some 10 proteins. To investigate the extent of conservation of the CIA pathway, we examined its importance in the early‐branching eukaryote Trypanosoma brucei that encodes all known CIA factors. Upon RNAi‐mediated ablation of individual, early‐acting CIA proteins, no major defects were observed in both procyclic and bloodstream stages. In contrast, parallel depletion of two CIA components was lethal, and severely diminished cytosolic aconitase activity lending support for a direct role of the CIA proteins in cytosolic Fe/S protein biogenesis. In support of this conclusion, the T. brucei CIA proteins complemented the growth defects of their respective yeast CIA depletion mutants. Finally, the T. brucei CIA factor Tah18 was characterized as a flavoprotein, while its binding partner Dre2 functions as a Fe/S protein. Together, our results demonstrate the essential and conserved function of the CIA pathway in cytosolic Fe/S protein assembly in both developmental stages of this representative of supergroup Excavata.     

Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target

The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100 μM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target.     

Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system

The flagellum of Trypanosoma brucei is a multifunctional organelle with critical roles in motility and other aspects of the trypanosome life cycle. Trypanin is a flagellar protein required for directional cell motility, but its molecular function is unknown. Recently, a trypanin homologue in Chlamydomonas reinhardtii was reported to be part of a dynein regulatory complex (DRC) that transmits regulatory signals from central pair microtubules and radial spokes to axonemal dynein. DRC genes were identified as extragenic suppressors of central pair and/or radial spoke mutations. We used RNA interference to ablate expression of radial spoke (RSP3) and central pair (PF16) components individually or in combination with trypanin. Both rsp3 and pf16 single knockdown mutants are immotile, with severely defective flagellar beat. In the case of rsp3, this loss of motility is correlated with the loss of radial spokes, while in the case of pf16 the loss of motility correlates with an aberrant orientation of the central pair microtubules within the axoneme. Genetic interaction between trypanin and PF16 is demonstrated by the finding that loss of trypanin suppresses the pf16 beat defect, indicating that the DRC represents an evolutionarily conserved strategy for dynein regulation. Surprisingly, we discovered that four independent mutants with an impaired flagellar beat all fail in the final stage of cytokinesis, indicating that flagellar motility is necessary for normal cell division in T. brucei. These findings present the first evidence that flagellar beating is important for cell division and open the opportunity to exploit enzymatic activities that drive flagellar beat as drug targets for the treatment of African sleeping sickness.     

DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair

DNA damage formation and repair are tightly linked to protein–DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication (ARS1). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct interactions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.     

Mycobacterial UvrD1 is a Ku-dependent DNA helicase that plays a role in multiple DNA repair events, including double-strand break repair

Mycobacterium tuberculosis and other bacterial pathogens have a Ku-dependent nonhomologous end joining pathway of DNA double-strand break repair. Here we identify mycobacterial UvrD1 as a novel interaction partner for Ku in a genome-wide yeast two-hybrid screen. UvrD1 per se is a vigorous DNA-dependent ATPase but a feeble DNA helicase. Ku stimulates UvrD1 to catalyze ATP-dependent unwinding of 3'-tailed DNAs. UvrD1, Ku, and DNA form a stable ternary complex in the absence of ATP. The Ku binding determinants are located in the distinctive C-terminal segment of UvrD1. A second mycobacterial paralog, UvrD2, is a vigorous Ku-independent DNA helicase. Ablation of UvrD1 sensitizes Mycobacterium smegmatis to killing by ultraviolet and ionizing radiation and to a single chromosomal break generated by I-SceI endonuclease. The physical and functional interactions of bacterial Ku and UvrD1 highlight the potential for cross-talk between components of nonhomologous end joining and nucleotide excision repair pathways.     

TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance

The trypanosome mitochondrial genome, kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings, including several thousand minicircles and dozens of maxicircles. The unusual complexity of kDNA would indicate that numerous proteins must be involved in its condensation, replication, segregation and gene expression. During our investigation of trypanosome mitochondrial PIF1-like helicases, we found that TbPIF8 is the smallest and most divergent. It lacks some conserved helicase domains, thus implying that unlike other mitochondrial PIF1-like helicases, this protein may have no enzymatic activity. TbPIF8 is positioned on the distal face of kDNA disk and its localization patterns vary with different kDNA replication stages. Stem-loop RNAi of TbPIF8 arrests cell growth and causes defects in kDNA segregation. RNAi of TbPIF8 causes only limited kDNA shrinkage but the networks become disorganized. Electron microcopy of thin sections of TbPIF8-depleted cells shows heterogeneous electron densities in the kinetoplast disk. Although we do not yet know its exact function, we conclude that TbPIF8 is essential for cell viability and is important for maintenance of kDNA.     

An evolutionarily conserved coiled-coil protein implicated in polycystic kidney disease is involved in basal body duplication and flagellar biogenesis in Trypanosoma brucei

Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism.