Mutation Frequency Decline Research Articles

The Transcription-Repair Coupling Factor Mfd Prevents and Promotes Mutagenesis in a Context-Dependent Manner.

The mfd (mutation frequency decline) gene was identified by screening an auxotrophic Escherichia coli strain exposed to UV and held in a minimal medium before plating onto rich or minimal agar plates. It was found that, under these conditions, holding cells in minimal (nongrowth) conditions resulted in mutations that enabled cells to grow on minimal media. Using this observation as a starting point, a mutant was isolated that failed to mutate to auxotrophy under the prescribed conditions, and the gene responsible for this phenomenon (mutation frequency decline) was named mfd. Later work revealed that mfd encoded a translocase that recognizes a stalled RNA polymerase (RNAP) at damage sites and binds to the stalled RNAP, recruits the nucleotide excision repair damage recognition complex UvrA2UvrB to the site, and facilitates damage recognition and repair while dissociating the stalled RNAP from the DNA along with the truncated RNA. Recent single-molecule and genome-wide repair studies have revealed time-resolved features and structural aspects of this transcription-coupled repair (TCR) phenomenon. Interestingly, recent work has shown that in certain bacterial species, mfd also plays roles in recombination, bacterial virulence, and the development of drug resistance.

Chemical composition, genotoxicity and antigenotoxicity study of Artemisia herba-alba using the eye and wing SMART assay of Drosophila melanogaster

The purpose of this study was to determine the chemical composition and to evaluate the genotoxic and antigentoxic activites of the Moroccan Artemisia herba-alba ethanolic extract. The ethanolic extract of Artemisia herba-alba was analyzed by gas chromatography coupled with mass spectroscopy (GC-MS). The mutagenic and antimutagenic activities were evaluated using thesomatic mutation and recombination test (SMART) on Drosophila melanogaster. Methyl methanesulfonate (MMS) was used as the positive control.Twenty-one compounds of the Artemisia herba-alba ethanol extract were identified; the main monoterpenes identified are camphor, α-thujone and β-thujone. Ethanolic extract of Artemisia herba-alba did not show gentotoxicity at doses used in SMART Assays. In the co-treatment, a dose-dependent decrease in mutation frequency was observed for this plant. Ethanolic extract of Artemisia herba-alba at 1.5% demonstrated a marked decrease in MMS with a strong inhibitory effect (80%). The results demonstrate that the Artemisia herba-alba exerted a significant and potent anti-mutagenic activity in wing and eye-spot of D. melanogaster.

Tetanus Toxin Synthesis is Under the Control of A Complex Network of Regulatory Genes in Clostridium tetani.

Clostridium tetani produces a potent neurotoxin, the tetanus toxin (TeNT), which is responsible for an often-fatal neurological disease (tetanus) characterized by spastic paralysis. Prevention is efficiently acquired by vaccination with the TeNT toxoid, which is obtained by C. tetani fermentation and subsequent purification and chemical inactivation. C. tetani synthesizes TeNT in a regulated manner. Indeed, the TeNT gene (tent) is mainly expressed in the late exponential and early stationary growth phases. The gene tetR (tetanus regulatory gene), located immediately upstream of tent, encodes an alternative sigma factor which was previously identified as a positive regulator of tent. In addition, the genome of C. tetani encodes more than 127 putative regulators, including 30 two-component systems (TCSs). Here, we investigated the impact of 12 regulators on TeNT synthesis which were selected based on their homology with related regulatory elements involved in toxin production in other clostridial species. Among nine TCSs tested, three of them impact TeNT production, including two positive regulators that indirectly stimulate tent and tetR transcription. One negative regulator was identified that interacts with both tent and tetR promoters. Two other TCSs showed a moderate effect: one binds to the tent promoter and weakly increases the extracellular TeNT level, and another one has a weak inverse effect. In addition, CodY (control of dciA (decoyinine induced operon) Y) but not Spo0A (sporulation stage 0) or the DNA repair protein Mfd (mutation frequency decline) positively controls TeNT synthesis by interacting with the tent promoter. Moreover, we found that inorganic phosphate and carbonate are among the environmental factors that control TeNT production. Our data show that TeNT synthesis is under the control of a complex network of regulators that are largely distinct from those involved in the control of toxin production in Clostridium botulinum or Clostridium difficile.

The Human RAD5 Homologs, HLTF and SHPRH, Have Separate Functions in DNA Damage Tolerance Dependent on The DNA Lesion Type.

Helicase-like transcription factor (HLTF) and SNF2, histone-linker, PHD and RING finger domain-containing helicase (SHPRH), the two human homologs of yeast Rad5, are believed to have a vital role in DNA damage tolerance (DDT). Here we show that HLTF, SHPRH and HLTF/SHPRH knockout cell lines show different sensitivities towards UV-irradiation, methyl methanesulfonate (MMS), cisplatin and mitomycin C (MMC), which are drugs that induce different types of DNA lesions. In general, the HLTF/SHPRH double knockout cell line was less sensitive than the single knockouts in response to all drugs, and interestingly, especially to MMS and cisplatin. Using the SupF assay, we detected an increase in the mutation frequency in HLTF knockout cells both after UV- and MMS-induced DNA lesions, while we detected a decrease in mutation frequency over UV lesions in the HLTF/SHPRH double knockout cells. No change in the mutation frequency was detected in the HLTF/SHPRH double knockout cell line after MMS treatment, even though these cells were more resistant to MMS and grew faster than the other cell lines after treatment with DNA damaging agents. This phenotype could possibly be explained by a reduced activation of checkpoint kinase 2 (CHK2) and MCM2 (a component of the pre-replication complex) after MMS treatment in cells lacking SHPRH. Our data reveal both distinct and common roles of the human RAD5 homologs dependent on the nature of DNA lesions, and identified SHPRH as a regulator of CHK2, a central player in DNA damage response.

Abstract 443: Blood-based genotyping and clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib in real-world settings: Results from POLARIS

Abstract Background:POLARIS is a prospective, real-world (RW) study of palbociclib (PAL) in patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) in the US and Canada. Analyses include evaluating the association between circulating tumor DNA (ctDNA) and clinical response to PAL. Methods: All pts provided informed consent. Blood samples are collected on day 1 of each 4-wk cycle for 6 cycles, at day 1 of every 2-3 cycles, and end of treatment. ctDNA is sequenced using the Guardant360 platform for somatic single-nucleotide variants in complete or critical exons of 73 genes. Analyses include association between clinical outcomes and ctDNA mutation frequencies at baseline and change from baseline to cycle 2, day 1 (C2D1), stratified by line of prior therapy. Results: As of July 2018, 127 pts had ≥1 evaluable ctDNA measurement. Of these, 104 had baseline and C2D1 results; 96 had tumor response assessed. The most common mutations at baseline were PIK3CA (41%), TP53 (33%), and ESR1 (20%). PAL was associated with a decline in mutation frequency of PIK3CA, TP53, and ESR1alleles but not all genes, highlighting differential response based on genomic profile and clonal heterogeneity (Table). Overall, fast progressors (progression <6 mo, n=19) tended to have higher baseline ctDNA values and displayed smaller decreases in ctDNA at C2D1 than slow progressors (progression ≥6 mo, n=4). Conclusions: This study is among the first to provide RW data on blood-based genotyping. Interim data indicate that genomic architecture and longitudinal changes may be predictive and prognostic in pts with HR+/HER2– ABC receiving PAL. Updated data from 239 pts will be presented, including association between individual ctDNA mutation dynamics, clonal heterogeneity, and clinical outcomes (therapeutic response and progression-free survival). Pfizer; NCT03280303 Change from baseline in ctDNA mutation frequencyMutationOverall (n=104)*First Line (n=70)Second Line and Later (n=34)Baseline, n (%)†C2D1, n (%)†Baseline, n (%)†C2D1, n (%)†Baseline, n (%)†C2D1, n (%)†PIK3CA43 (41)26 (25)27 (39)14 (20)16 (47)12 (35)TP5334 (33)27 (26)23 (33)17 (24)11(32)10 (29)ESR121 (20)11 (11)9 (13)3 (4)12 (35)8 (24)ARID1A19 (18)14 (13)13 (19)7 (10)6 (18)7 (21)NF111 (11)11 (11)7 (10)6 (9)4 (12)5 (15)BRCA210 (10)10 (10)8 (11)7 (10)2 (6)3 (9)GATA310 (10)5 (5)8 (11)5 (7)2 (6)O (O)None detected13 (13)24 (23)7 (10)20 (29)6 (18)4 (12)ctDNA=circulating tumor DNA; C2D1=cycle 2, day 1. *Patients with both baseline and C2D1 ctDNA data available. †Percentages are relative to the corresponding number of patients (n). Citation Format: Aditya Bardia, Joanne L. Blum, Steven L. McCune, Kamal Patel, Richard C. Frank, Kailash Mosalpuria, Meghan S. Karuturi, Lloyd A. Shabazz, Gabrielle B. Rocque, Yuan Liu, Zhe Zhang, Jian Wang, Yao Wang, Debu Tripathy. Blood-based genotyping and clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib in real-world settings: Results from POLARIS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 443.

Non-canonical processing of DNA photodimers with Bacillus subtilis UV-endonuclease YwjD, 5′→3′ exonuclease YpcP and low-fidelity DNA polymerases YqjH and YqjW

It has been shown that mutation frequency decline (Mfd) and nucleotide excision repair (NER) deficiencies promote UV-induced mutagenesis in B. subtilis sporangia. As replication is halted in sporulating B. subtilis cells, in this report, we investigated if this response may result from an error-prone repair event involving the UV-endonuclease YwjD and low fidelity (LF) DNA synthesis. Accordingly, disruption of YwjD generated B. subtilis sporangia that were more susceptible to UV-C radiation than sporangia of the WT strain and such susceptibility increased even more after the single or simultaneous inactivation of the LF DNA polymerases YqjH and YqjW. To further explore this concept, functional His6-tagged YwjD and Y-DNA polymerases YqjH and YqjW were produced and purified to homogeneity. In vitro repair assays showed that YwjD hydrolyzed the phosphodiester bond immediately located 5´-end of a ds-DNA substrate bearing either, cyclobutane pyrimidine dimers (CPD), 6-4 photoproducts (6-4 PD) or Dewar isomers (DWI). Notably, the 6-4 PD and DWI but not the CPDs repair intermediaries of YwjD were efficiently processed by the LF polymerase YqjH suggesting that an additional 5′→3′ exonuclease event was necessary to process PD. Accordingly, the LF polymerase YqjW efficiently processed the incision-excision repair products derived from YwjD and exonuclease YpcP attack over CPD-containing DNA. In summary, our results unveiled a novel non-canonical repair pathway that employs YwjD to incise PD-containing DNA and low fidelity synthesis contributing thus to mutagenesis, survival and spore morphogenesis in B. subtilis.

MFD Dynamically Regulates Transcription

Fundamental processes that occur along DNA, such as transcription and replication, commonly encounter obstacles or 'roadblocks' which may impede progress and ultimately result in mutations and/or DNA damage. Thus, spatiotemporal control of these factors can have far-reaching consequences. In bacteria, the mutation frequency decline (Mfd) protein has been widely recognized for dealing with transcriptional roadblock resolution through either facilitating transcription through a roadblock or terminating transcription altogether. Despite this importance, it is unclear how Mfd interacts with RNAP and DNA and how such interactions lead to obstacle resolution. Using novel assays, we discovered that Mfd interacts with RNAP in a dynamic and highly regulated fashion. Our findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd, and expedient conflict resolution.

Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 μg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 μg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

The Bacterial Mfd Protein Prevents DNA Damage Induced by the Host Nitrogen Immune Response in a NER-Independent but RecBC-Dependent Pathway.

Production of reactive nitrogen species is an important component of the host immune defence against bacteria. Here, we show that the bacterial protein Mfd (Mutation frequency decline), a highly conserved and ubiquitous bacterial protein involved in DNA repair, confers bacterial resistance to the eukaryotic nitrogen response produced by macrophage cells and during mice infection. In addition, we show that RecBC is also necessary to survive this stress. The inactivation of recBC and mfd genes is epistatic showing that Mfd follows the RecBC repair pathway to protect the bacteria against the genotoxic effect of nitrite. Surprisingly given the role of Mfd in transcription-coupled repair, UvrA is not necessary to survive the nitrite response. Taken together, our data reveal that during the eukaryotic nitrogen response, Mfd is required to maintain bacterial genome integrity in a NER-independent but RecBC-dependent pathway.

Deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype 1b hepatitis C virus.

Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy. In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.

Morphoanatomic characterization of the stem and the leaf of Tabernaemontana catharinensis A.DC (Apocynaceae) and antimutagenic activity of its leaves

ABSTRACT Tabernaemontana catharinensis A. DC (Apocynaceae) is used as a medicinal plant by the population. In order to contribute to the safe use of the plant as herbal medicine, this study aimed to morphoanatomically characterize the aereal vegetative organs of T. catharinensis and to evaluate the leaves’ mutagenic and antimutagenic activities. Histological blades of leaves and stem of T. catharinensis were performed; the methionine system (methG1) and Aspergillusnidulans conidia germination analysis were employed for mutagenic and antimutagenic evaluation. The morphoanatomic analysis did not show trichomes in the stem, petiole and leaf. Besides, it was observed both the presence of bi-collateral bundles - except in the foliar apex where the bundles were from the collateral type - as well as anamphistomatic leaf with paracyte stomata and sub-epidermal layer in the region of the leaf edges. The mutagenicity/antimutagenicity trial indicated a significant decrease of mutation frequency in comparison with the control group and showed that the T. catharinensis had antimutagenic activity within the type, time and form of treatment. Since the germination test showed that the conidia germination was accelerated from the bud phase, activities at the cell cycle level and polarized growth proved to be possible. The morphoanatomic analysis of the leaf and stem associated with the mutagenic and antimutagenic analyses contributes to the safe use of the plant by humans and also for the quality control of a possible phytotherapeutic drug.

Transcriptional pausing to scout ahead for DNA damage

A cell’s genome is under constant threat of damage, which if not repaired can lead to mutations or cell death. Common forms of DNA damage found in nature include cyclobutane pyrimidine dimers and 6-4 photoproducts induced by UV-irradiation. These and other helix-distorting lesions are removed by a highly conserved process called nucleotide excision repair (NER) that is found in every kingdom of life (1). NER is initiated in two general ways: by damage recognition proteins that survey the entire genome for damage or lesion-induced transcriptional stalling. This latter pathway, called transcription-coupled repair (TCR), first reported in mammalian cells and then in bacteria, is initiated when RNA polymerase (RNAP) is arrested at a DNA lesion embedded in the transcribed strand (2, 3). However, before DNA repair enzymes obtain access, the stalled RNAP must be pushed away from the lesion by the action of DNA translocases. Thus, the repair “coupling factors,” which recognize the stalled RNAP, must work to both displace the polymerase and simultaneously enlist the repair proteins to remove the damage. In bacteria, two different TCR pathways have emerged involving two different DNA helicases, which help to displace RNAP. The Mfd (mutation frequency decline) protein, also called transcription-repair coupling factor, uses its helicase fold and ATP hydrolysis to literally push RNAP forward (downstream) past the damaged site (Fig. 1A) (reviewed in ref. 4), whereas in a newly discovered alternative pathway UvrD (helicase II) tows the RNAP backward (upstream) with the help of the transcription elongation factor, NusA (5). This second approach more closely resembles what is thought to occur in mammalian cells during TCR (6). As described below, Mfd targets the nucleotide excision repair system to sites of damage through its direct interaction with a stalled RNAP. However, nature has gone even further in devising ways to find and remove potentially RNAP-blocking DNA damage. As described by Haines et al. (7) in PNAS, Nigel Savery’s group at the University of Bristol have found that the Mfd protein that normally accompanies the translocating RNAP can be sent ahead of a blocked RNAP to scout for damage in the transcribed strand and facilitate the recruitment of the bacterial NER machinery.

English

The antigenotoxic action of fennel (Foeniculum vulgare) were assessed using the eye (w/w+) somatic mutation and recombination test (SMART) assay of Drosophila melanogaster. Fennel is used for various medicinal purposes. Methyl methanesulfonate (MMS) was used as the positive control. Fruit extracts of fennel did not show genotoxicity at the doses used. In the co-treatment, a dose-dependent decrease in mutation frequency was observed for this plant. The fennel at 8% (w/v) demonstrated a marked decrease in MMS with an inhibition rate of 41.16%. The results demonstrate that the fennel exerted a significant and potent anti-mutagenic activity in eyes spot of D. melanogaster. Key words: Drosophila melanogaster, somatic mutation and recombination test (SMART), antigenotoxicity, Foeniculum vulgare, methyl methanesulfonate (MMS).

The effect of calorie restriction on the genome stability in DNA repair defective cells

It has been found that calorie restriction (CR) can extend the average and maximum life span from yeast to primates and delay the onset of age‐associated pathologies. Mismatch repair and double strand break repair systems are major DNA repair pathways whose function are critical for maintaining genome stability. The inactivation of these systems in human pathway has been reported to cause cancer predisposition such as Hereditary Nonpolyposis Colorectal Cancer, Werner and Bloom syndromes. By using Saccharomyces cerevisiae model system for CR and aging studies, DNA repair defective cells (msh2Δ, msh3Δ, msh6Δ and sgs1Δ) were cultured with normal (2% glucose) and CR (0.5% glucose) medium. Genome stability studies were analyzed by CANr, Homr and Lysr mutation assay. Results show that CR can significantly extend the lifespan of all wild‐type and mutants. Besides, CR shows significant decrease in HOMr mutation frequency by 2.7 × 104‐fold in msh2Δ mutant, whereas by 2.4 × 106‐fold and 1.4 × 106‐fold reduction in msh3Δ mutant for HOMr and LYSr assay respectively. In addition, for sgs1Δ mutant, there are significant decreases in HOMr and LYSr assay by two‐fold. Under normal condition, msh2Δ, msh3Δ and sgs1Δ cells found to have regrow phenotype upon aging. However, this phenotype was diminished under CR condition. Thus, CR promotes genome stability in DNA repair defective cells especially in msh2Δ, msh3Δ and sgs1Δ mutants.

Analysis of mice deficient in both REV1 catalytic activity and POLH reveals an unexpected role for POLH in the generation of C to G and G to C transversions during Ig gene hypermutation

Multiple DNA polymerases are involved in the generation of somatic mutations during Ig gene hypermutation. Mice expressing a catalytically inactive REV1 (REV1AA) exhibit reduction of both C to G and G to C transversions and moderate decrease of A/T mutations, whereas DNA polymerase η (POLH) deficiency causes greatly reduced A/T mutations. To investigate whether REV1 and POLH interact genetically and functionally during Ig gene hypermutation, we established REV1AA Polh(-/-) mice and analyzed Ig gene hypermutation in the germinal center (GC) B cells. REV1AA Polh(-/-) mice were born at the expected ratio and developed normally with no apparent gross abnormalities. B-cell development, maturation, Ig gene class switch and the GC B-cell expansion were not affected in these mice. REV1AA Polh(-/-) B cells also exhibited relatively normal sensitivity to etoposide and ionizing radiation. Analysis of somatic mutations in the J(H)4 intronic region revealed that REV1AA Polh(-/-) mice had a further decrease of overall mutation frequency compared with REV1AA or Polh(-/-) mice, indicating that the double deficiency additively affected the generation of mutations. Remarkably, REV1AA Polh(-/-) mice had nearly absent C to G and G to C transversions, suggesting that POLH is essential for the generation of residual C to G and G to C transversions observed in REV1AA mice. These results reveal genetic interactions between REV1 catalytic activity and POLH and identify an alternative pathway, mediated by non-catalytic REV1 and POLH, in the generation of C to G and G to C transversions.

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G>C≫A>T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA−Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.Mutation spectrum established for strains expressing only Pol V, showed that in uvrA− bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T→C:G, A:T→G:C, G:C→A:T and G:C→T:A prevailed.The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.

Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor

Transcription coupled nucleotide excision repair (TC-NER) is involved in correcting UV-induced damage and other road-blocks encountered in the transcribed strand. Mutation frequency decline (Mfd) is a transcription repair coupling factor, involved in repair of template strand during transcription. Mfd from M. tuberculosis (MtbMfd) is 1234 amino-acids long harboring characteristic modules for different activities. Mtbmfd complemented Escherichia coli mfd (Ecomfd) deficient strain, enhanced survival of UV irradiated cells and increased the road-block repression in vivo. The protein exhibited ATPase activity, which was stimulated ∼1.5-fold in the presence of DNA. While the C-terminal domain (CTD) comprising amino acids 630 to 1234 showed ∼2-fold elevated ATPase activity than MtbMfd, the N-terminal domain (NTD) containing the first 433 amino acid residues was able to bind ATP but deficient in hydrolysis. Overexpression of NTD of MtbMfd led to growth defect and hypersensitivity to UV light. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC) increased the ATPase activity by ∼10-fold and correspondingly exhibited efficient translocation along DNA as compared to the MtbMfd and CTD. Surprisingly, MtbMfd was found to be distributed in monomer and hexamer forms both in vivo and in vitro and the monomer showed increased susceptibility to proteases compared to the hexamer. MfdΔC, on the other hand, was predominantly monomeric in solution implicating the extreme C-terminal region in oligomerization of the protein. Thus, although the MtbMfd resembles EcoMfd in many of its reaction characteristics, some of its hitherto unknown distinct properties hint at its species specific role in mycobacteria during transcription-coupled repair.

Correlation of polymerase replication fidelity with genetic evolution of influenza A/Fujian/411/02(H3N2) viruses

Influenza virus evolves continuously through mutations presumed to result from evolutionary pressure driving viral replication. This study examined the relationship between the genetic evolution and replication fidelity of influenza viruses. Analysis of influenza sequences from National Centre for Biotechnology Information (NCBI) database revealed a gradual decrease in the rate of genetic evolution of A/Fujian/411/02(H3N2)-like variants after the emergence and predominance of the A/H3N2 Fujian strain in 2002. This decrease may be related to an increase in replication fidelity, which was investigated by assessing mutation frequencies of reassortant viruses carrying the PB1 segment of Fujian variants isolated between 2003 and 2005 in a sequencing-based plaque assay. The data revealed a threefold decrease in substitution per site of the reassortant viruses carrying the Fujian PB1 segments isolated in 2004-2005 compared with those circulating in 2003. The decrease in mutation frequency paralleled a decrease in genetic evolution of the Fujian variants from the NCBI database. This correlation implicates changes in the polymerase replication fidelity as contributing to altered genetic evolution of influenza viruses.

Regulation and Rate Enhancement during Transcription-Coupled DNA Repair

SummaryTranscription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) that is triggered when RNA polymerase is stalled by DNA damage. Lesions targeted by TCR are repaired more quickly than lesions repaired by the transcription-independent “global” NER pathway, but the mechanism underlying this rate enhancement is not understood. Damage recognition during bacterial NER depends upon UvrA, which binds to the damage and loads UvrB onto the DNA. Bacterial TCR additionally requires the Mfd protein, a DNA translocase that removes the stalled transcription complexes. We have determined the properties of Mfd, UvrA, and UvrB that are required for the elevated rate of repair observed during TCR. We show that TCR and global NER differ in their requirements for damage recognition by UvrA, indicating that Mfd acts at the very earliest stage of the repair process and extending the functional similarities between TCR in bacteria and eukaryotes.

BRCA1 gene mutations may explain more than 80% of excess number of ovarian cancer cases after breast cancer – a population based study from the Western Sweden Health Care region

Aim. In a previous cohort study, we showed that there was a significant variation in the frequency of ovarian cancer after having breast cancer in Sweden, with the highest risk occuring in the Western region. The present study aimed to evaulate whether the high prevalence of the founder mutation BRCA1 3171ins5 may explain the excess number of ovarian cancer. Method. Among more than 26 000 women with breast cancer in the Western Swedish Health Care Region, 159 cases were subsequently diagnosed with ovarian cancer, whereas the expected number was 96. Archived tissue material was analysed for six common Scandinavian BRCA1 and BRCA2 gene mutations. Results. The excess number of cases was 63 (95% CI 47–77), based on person-years at risk and national incidence rates of ovarian cancer. A BRCA1 gene mutation was detected in 33 cases corresponding to 52% of the excess number. The founder mutation, BRCA1 3171ins5, was detected in 44% of the excess number. The identified mutations decreased from 45% in women less than 50 years of age at follow-up to 14% at 60+ years at follow-up. There was no obvious decrease in mutation frequency by excess numbers with age. Age at follow-up and first-degree relatives with breast and/or ovarian cancer were the best predictors of a mutation in this material. Conclusion. The founder mutation, BRCA1 3171ins5, explains the excess of ovarian cancer after breast cancer in the region. From the relative frequency of the studied mutations found at the cancer genetic counselling clinic, it is estimated that BRCA1 gene mutations are associated with about 80–85% of the excess cases. This means that a negative screening for these mutations in similar cases may have a predictive value and could strongly reduce the risk of ovarian cancer in relatives.