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PubMed2003

Atazanavir.

Atazanavir is a novel azapeptide protease inhibitor with high specificity for, and activity against, HIV-1 protease. The resistance profile of atazanavir is distinct, with an I50 L protease substitution appearing to be the signature mutation. Atazanavir was not associated with increases in total cholesterol, low density lipoprotein-cholesterol or triglyceride levels after 108 weeks. Atazanavir has a pharmacokinetic profile that allows for once-daily oral administration. It is a moderate inhibitor of hepatic cytochrome P450 enzymes and interacts with several drugs. In combination with stavudine plus didanosine, atazanavir 200, 400 or 500 mg once daily produced a rapid and sustained reduction from baseline in viral load of 2.57, 2.42 and 2.53 log(10) copies/mL, respectively, in treatment-naive patients after 48 weeks, compared with a decrease of 2.33 log(10) copies/mL with nelfinavir 750 mg three times daily. Nausea was the most clinically relevant adverse event reported in patients receiving atazanavir-based regimens.

Administration, Oral↗
PubMed2026

Long-Read Haplotype Phasing Resolves Allelic Configuration as a Missing Layer of Precision Oncology.

Conventional short-read sequencing cannot determine whether co-occurring variants within a cancer gene reside on the same allele (cis) or on opposing alleles (trans), a distinction with direct biological and therapeutic consequences. Trans configurations confirm biallelic tumor suppressor inactivation and inform therapy selection, while cis configurations generate compound oncogenic alleles with enhanced activity. We analyzed 768 patients with prostate, breast, or ovarian cancers in the PROBLEM cohort, using mutational signatures to nominate cryptic genomic instability cases where the causative biallelic event was not apparent from short-read sequencing. Long-read nanopore sequencing resolved 32 of 46 cryptic cases (69.6%), leveraging its unique advantages in direct methylation detection, long insertion resolution, and complex structural variant characterization, confirming trans biallelic inactivation in all resolved tumor suppressor cases. Systematic analysis of 4,496 MiOncoSeq samples identified 17,519 multi-hit gene pairs, of which 78.7% exceeded the 500 bp short-read phasing limit. Long-read phasing further revealed recurrent compound cis oncogenic alleles in NOTCH1, PIK3CA, PDGFRB, and KIT with functionally synergistic activity. Haplotype phasing resolves a systematically overlooked gap in cancer variant interpretation and warrants broader integration into precision oncology workflows.

Journal Article↗
PubMed1999

Simultaneous phenotypically distinct but clonally identical mucosa-associated lymphoid tissue and follicular lymphoma in a patient with Sjögren's syndrome.

A 44-year-old woman with a 12-year history of Sjögren's syndrome (SS) developed a low-grade mucosa-associated lymphoid tissue (MALT) lymphoma in the parotid gland. Two years later, she presented with generalized lymphadenopathy and hepatosplenomegaly and a follicular lymphoma was diagnosed. To investigate the relationship of the two histologically distinct lymphomas, we re-examined their histology and immunophenotype and studied the lymphomatous tissue from the parotid, cervical lymph node, and spleen using molecular genetic methods. Histologic and immunophenotypic studies confirmed the previous diagnoses and also identified a previously unnoticed focus of follicular lymphoma in the second parotid gland biopsy. Polymerase chain reaction (PCR) amplification of the rearranged Ig heavy-chain gene showed the same sized dominant product in the MALT lymphoma and the follicular lymphoma. Similarly, PCR analysis of the t(14:18) translocation yielded an identical sized band from both MALT and follicular lymphoma. Cloning and sequencing of the Ig PCR products showed an identical CDR3 sequence from each lesion, indicating a common clonal lineage. The follicular lymphoma of the parotid gland lymph node and the follicular lymphoma of the spleen showed an identical mutation signature to that of the salivary gland MALT lymphoma. We propose that follicular lymphoma in the parotid gland lymph node may have resulted from colonization of lymphoid follicles by MALT lymphoma cells, following which the tumor cells were induced to express a follicular lymphoma phenotype, due to Bcl-2 overexpression caused by t(14;18), leading to a change in clinical behavior resulting in rapid widespread dissemination of disease. These observations suggest that the distinct phenotypes of low-grade B-cell lymphomas may be the consequence of interplay between genetic and local microenvironmental factors.

Adult↗
PubMed2004

Biological consequences of 8-methoxypsoralen-photoinduced lesions: sequence-specificity of mutations and preponderance of T to C and T to a mutations.

Psoriatic patients undergoing psoralen plus ultraviolet radiation (PUVA) therapy are susceptible for squamous cell carcinoma and melanoma of the skin. To investigate the etiological relevance of PUVA for these diseases, we performed mutation spectrometry on the cII transgene in mouse embryonic fibroblasts treated with a single or split PUVA dose (PUVA-I or PUVA-II, respectively). Both treatments were significantly mutagenic as they increased the cII mutant frequency up to 3.7-fold over background, and produced different mutational spectra from that derived spontaneously (p<0.01), but not from one another. The signature of induced mutations, i.e., T to C transitions and T to A transversions with significant site-specificities, i.e., adjacent to T bases at the 3'-neighboring side and to pyrimidines at the 5'-neighboring side, was more pronounced after PUVA-II treatment. Also, the overall mutations occurring at T bases with the same site-specificities were more prevalent after PUVA-II treatment. The characteristic PUVA-induced mutations predominate in the p53 mutational spectrum in controlled in vivo test systems or in high-dose PUVA-treated patients, and also are easily recognizable in the overall PUVA-treated patients. We conclude that PUVA-induced mutagenesis is initiated by PUVA-I treatment and subsequently, augmented by PUVA-II treatment, leaving a unique mutational signature on the cII transgene. The signature mutations of PUVA are discernible in the p53 mutational spectrum in PUVA-treated patients but complex exposure to other therapeutic/environmental carcinogens also leads to the frequent occurrence of other types of mutations in this population.

Animals↗
PubMed2004

Identification of I50L as the signature atazanavir (ATV)-resistance mutation in treatment-naive HIV-1-infected patients receiving ATV-containing regimens.

Atazanavir (ATV) is a once-daily human immunodeficiency virus (HIV) protease inhibitor (PI) shown to be effective and well tolerated. ATV has a distinct resistance profile relative to other PIs, with susceptibility maintained against 86% of isolates resistant to 1-2 PIs. Clinical isolates obtained from PI-naive patients designated as experiencing virologic failure while receiving ATV-containing regimens contained a unique isoleucine-to-leucine substitution at amino acid residue 50 (I50L) of the HIV-1 protease. The I50L substitution, observed in all isolates exhibiting phenotypic resistance to ATV, emerged in a variety of different backgrounds and was most frequently accompanied by A71V, K45R, and/or G73S. Viruses containing an I50L substitution were growth impaired, displayed ATV-specific resistance, and had increased susceptibilities (</=0.4 of reference strain) to other PIs. Comparison of viruses bearing I50L with those bearing I50V revealed specific resistance to ATV and amprenavir, respectively, with no evidence of cross-resistance. The unique I50L substitution is the signature mutation for resistance to ATV.

Atazanavir Sulfate↗
PubMed2003

Detection of cystic fibrosis mutations by peptide mass signature genotyping.

BACKGROUND: The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis. METHODS: We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149-54) were assessed using the PMSG test for the CFTR gene. RESULTS: Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing. CONCLUSIONS: The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.

Cloning, Molecular↗
PubMed2003

Enhancement of the mutagenicity of benzo(a)pyrene diol epoxide by a nonmutagenic dose of ultraviolet A radiation.

We investigated the effects of single and combined exposures to two ubiquitous environmental carcinogens, polycyclic aromatic hydrocarbons and UVA radiation, in Big Blue mouse embryonic fibroblasts. We quantified the cytotoxicity, DNA adduct formation, and induction of mutations in the cII transgene in cells treated with a single agent or combinations of agents in both direct and reverse order. Mapping of DNA adducts by terminal transferase-dependent PCR showed the preferential formation of bulky adducts at identical nucleotide positions along the cII gene after treatment with the prototype polycyclic aromatic hydrocarbon, benzo(a)pyrene diol epoxide [B(a)PDE], or B(a)PDE plus UVA radiation treatments but not after UVA irradiation alone. The cII mutant frequency determined by a lambda phage-based mutation detection system was not increased significantly by UVA irradiation (1.7-fold over background; P < 0.3); however, B(a)PDE alone or in combinations with UVA radiation significantly increased the cII mutant frequency (P < 0.001). The highest cII mutant frequency was induced by the treatment with B(a)PDE followed by UVA irradiation, which was more than the added mutant frequencies of the two agents individually (>12.2-fold versus <7.6-fold over background; P < 0.01). In support of these findings, DNA sequencing analyses showed that the mutational spectra induced by B(a)PDE alone or combined with UVA radiation were significantly different from those derived spontaneously (P < 0.0001) or by UVA irradiation (P < 0.0005). The signature of mutations produced by B(a)PDE i.e., "G-->T + C-->A" transversions, was significantly enhanced when the B(a)PDE treatment was followed by UVA irradiation (47% versus 65%; P < 0.01). Also, the methylated CpG dinucleotide-targeted overall mutations specifically induced by B(a)PDE were increased after the subsequent UVA irradiation (43% versus 51%, respectively). Such enhancements in the mutational signature of B(a)PDE were most pronounced within the preferential DNA adduction sites along the cII gene after the treatment with B(a)PDE plus UVA radiation, which suggests that the primary B(a)PDE adducts are converted to more mutagenic species on UVA irradiation. We conclude that UVA radiation at a nonmutagenic dose has an enhancing effect on the mechanism by which B(a)PDE induces mutations.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2005

Analysis of the lineage relationship between mast cells and basophils using the c-kit D816V mutation as a biologic signature.

BACKGROUND: Mast cells and basophils share similar morphologic and functional properties; however, it is not known whether they are derived from a bilineage (basophil/mast cell)-restricted progenitor. OBJECTIVE: To assess whether basophils and mast cells are derived from common committed progenitors using the c-kit D816V mutation as a biologic signature. METHODS: The D816V c-kit mutation found in mast cells of patients with systemic mastocytosis is used as a trackable genetic marker to assess the lineage relationship between mast cells and basophils. Blood and bone marrow aspirates were collected from 33 consecutive patients with mastocytosis with different disease severity. Peripheral blood basophils, monocytes and neutrophils were sorted by immunomagnetic beads. Presence of the D816V c-kit mutation was analyzed by restriction fragment length polymorphism in the genomic DNA and mRNA from sorted cells in all patients and in the genomic DNA of individual basophils of 1 patient. RESULTS: The c-kit D816V mutation was detectable in basophils of 5 patients (15%). All 5 patients had the c-kit mutation also detectable in monocytes and thus had multilineage involvement. Single cell analysis of the genomic DNA in 1 patient showed a similar degree of clonal expansion in basophils, monocytes, and neutrophils. Mutated c-kit was expressed at the mRNA level in all 5 patients. There was no difference in surface Kit expression levels in basophils. CONCLUSION: Basophils carrying the D816V c-kit mutation in mastocytosis were detected only in the context of a multilineage involvement. These results argue against the presence of a bilineage-restricted committed progenitor for mast cells and basophils.

Basophils↗
PubMed2004

The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein.

Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. The class III CF mutations G551D and G1349D are located within the "signature" sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Similar half-maximal effective concentrations (EC(50)) of MPB-91 (22-36microM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. The isoflavone genistein stimulates wild-type (wt)- and delF508-CFTR channel activity in a non-Michaelis-Menten manner. By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. We conclude from these results that whereas G551 in NBD1 is an important molecular site for inhibition of CFTR by genistein, the symmetrical G1349 in NBD2 is also one major site but for the activation of CFTR by genistein. Because both mutations alter specifically the mechanism of CFTR channel activation by genistein, we believe that the signature sequences of CFTR act as molecular switches that upon interaction with genistein turn on and off the channel.

Amino Acid Motifs↗
PubMed2025

Comparative Analysis of Somatic and Germline Polymerase Proofreading Deficiencies in Cancer: Molecular and Clinical Implications.

Polymerases &#x3b5; and &#x3b4; maintain genome integrity through exonuclease proofreading. Germline and somatic pathogenic variants (PVs) in the exonuclease domain (ED) of POLE and POLD1 impair proofreading, causing hypermutated tumors. Despite shared mutational features that make these tumors highly immunogenic, molecular and clinical distinctions between POLE and POLD1 mutations and between somatic and germline variants remain incompletely understood. We compared the molecular and clinical characteristics of POLE and POLD1 ED PVs (n = 31), assessing their location, pathogenicity, clinical phenotypes, mismatch repair (MMR) status, tumor mutational burden, and signatures. We analyzed 360 proofreading-deficient tumors (source: The Cancer Genome Atlas [TCGA] and Catalogue Of Somatic Mutations In Cancer [COSMIC]) and 70 families (249 individuals) with polymerase proofreading-associated polyposis. All germline and somatic PVs had high AlphaMissense scores (0.87-1) and clustered within or near Exo motifs. Recurrent, nonfounder germline PVs, POLE L424V and POLD1 S478N, showed low/modest REVEL scores. Somatic variants occurred mainly in endometrial cancers (75% of proofreading-deficient TCGA cancers), whereas colorectal cancer predominated in polymerase proofreading-associated polyposis (56% of carriers). Cancer risks and tumor spectra differed between POLE and POLD1 PV carriers. Aggressive hereditary phenotypes were linked to either specific POLE PVs (eg, S297F, V411L, P436R, M444K, A456P, and S461T) or the co-occurrence of germline ED PVs with germline MMR gene PVs. Distinct hypermutator profiles were confirmed for polymerase &#x3b5; and polymerase &#x3b4; proofreading deficiencies via unique mutational signatures (Polymerase &#x3b5;: SBS10a/b, SBS28; Polymerase &#x3b4;: SBS10c/d). Tumors with combined proofreading and MMR deficiencies had significantly higher tumor mutational burden and a shift in the associated mutational spectra. Unlike POLE, POLD1 ED PVs exhibited haplosufficiency, typically requiring a somatic second hit (eg, loss of heterozygosity) or MMR deficiency to drive hypermutation. In conclusion, differences between POLE and POLD1 and between somatic and germline mutations influence clinical presentation, mutagenic potential, and reliance on cooperating defects in tumorigenesis. These insights advance the understanding of proofreading-deficient cancers, with implications for diagnostics, genetic counseling, and precision oncology.

Humans↗
PubMed2025

Defining the genome-wide mutagenic impact of APOBEC3 enzymes.

Somatic mutations drive cancer initiation and tumor evolution. Therefore, the etiology of mutagenesis in cancer is important to preventative and treatment strategies. Somatic mutagenesis in cancer is a multifactorial process and includes both endogenous and exogenous sources of mutations. One recently recognized source of mutagenesis in cancer is the innate immune APOBEC3 family of enzymes, which catalyze cytosine deamination to restrict viral infection but can aberrantly act on the cellular genome, resulting in mutations. Single base substitution (SBS) signatures, or mutational patterns, identified in cancer genomes have demonstrated widespread mutagenesis caused by APOBEC3 enzymes throughout human tumors. To comprehensively define the consequences of APOBEC3 mutagenesis, we developed an experimental pipeline for prospective analysis of genome-wide mutations caused by APOBEC3 activity. This pipeline can be adapted to analyze additional sources of mutagenesis across a spectrum of cells.

Humans↗
PubMed2004

Detection and assignment of CYP21 mutations using peptide mass signature genotyping.

Congenital adrenal hyperplasia (CAH) is a common inborn error of steroidogenesis. The clinical spectrum of CAH ranges from the severe classical form, which can be fatal in the newborn, to simple virilizing forms or a milder non-classical form which is often not diagnosed until puberty. Recessive mutations in the autosomal gene encoding 21-hydroxylase (CYP21) are responsible for approximately 95% of CAH cases. Since CYP21 genotype is generally predictive of the presence and severity of the disorder, accurate CYP21 genotyping is of clear medical significance. Determining the CYP21 genotype of an individual, using standard methods, is difficult due to the presence of a nearly identical pseudogene (CYP21P) in close proximity to the functional gene. To address the need for a comprehensive test for mutations in the CYP21 gene, we developed a multiplexed peptide mass signature genotyping (PMSG) assay and applied the assay to 151 DNA samples. CAH patients had been previously characterized for the 10 most common mutations. The PMSG assay detected all common mutations; in addition it identified six known rare mutations and also discovered four new mutations (two frameshifts in the first half of the gene, P42fs and S171fs, and two point mutations, H365Y and R479L). This assay has the potential to provide high-throughput, cost-effective analysis of the CYP21 gene to detect known mutations and identify novel variants in samples obtained from patients with CAH, individuals suspected to have CAH, and heterozygous carriers.

Adrenal Hyperplasia, Congenital↗
PubMed1999

Functional consequences of mutations in the conserved 'signature sequence' of the ATP-binding-cassette protein MalK.

The binding-protein-dependent maltose-transport system of enterobacteria, a member of the ATP-binding-cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. Isolated MalK displays spontaneous ATPase activity, whereas in the assembled MalFGK2 complex, reconstituted in liposomes, ATP hydrolysis requires stimulation by the substrate-loaded extracellular maltose-binding protein, MalE. The ATPase domains of ABC transporters, including MalK, share a unique sequence motif ('LSGGQ', 'signature sequence' or 'linker peptide') with as yet unknown function. To elucidate its role in the transport process, we investigated the consequences of mutations affecting two highly conserved residues (G137, Q140) in the MalK-ATPase of Salmonella typhimurium, by biochemical means. Residues corresponding to Q140 in other ABC proteins have not yet been studied. All mutant alleles (G137--> A, V, T; Q140--> L, K, N) fail to restore a functional transport complex in vivo. In addition, the mutations increase the repressing activity of MalK on other maltose-regulated genes when compared with wild-type MalK. Purified variants of G137 have lost the ability to hydrolyze ATP but still display nucleotide-binding activity, albeit with reduced affinity. Binding of MgATP results in similar protection against trypsin, as observed with wild-type, indicating no major change in protein structure. In contrast, the variants of Q140 differ in their properties, depending on the chemical nature of the replacement residue. MalKQ140L fails to hydrolyze ATP and exhibits a strong intrinsic resistance to trypsin in the absence of MgATP, suggesting a drastically altered conformation. In contrast, the purified mutant proteins Q140K and Q140N display ATPase activities and MgATP-induced changes in the tryptic cleavage pattern similar to those of wild-type. However, mutant transport complexes containing the Q140K or Q140N variants, when studied in proteoliposomes, are severely impaired in MalE-maltose-stimulated ATPase activity. These results are discussed with respect to the crystal structure of the homologous HisP protein [Hung, L.-W., Wang, I.X., Nikaido, K., Liu, P.-Q., Ames, G.F.-L. & Kim, S.-H. (1998) Nature (London) 396, 703-707] and are interpreted in favor of a role of the signature sequence in activating the hydrolyzing activity of MalK upon substrate-initiated conformational changes in MalF/MalG.

ATP-Binding Cassette Transporters↗
PubMed2006

Most spontaneous tumors in a mouse model of Li-Fraumeni syndrome do not have a mutator phenotype.

Mutations are the substrate of cancer. Yet, little is known about the degree and nature of mutations in tumors because measurement of mutation load in tumors and normal tissues was generally not possible until the advent of transgenic mouse mutation detection systems. Herein, we present the first analysis of mutation frequency and pattern in thymic tumors from a mouse model of Li-Fraumeni syndrome (p53+/- murine model) using the Big Blue assay with sequencing of all mutants. We also make the first characterization of mutation frequency and pattern in p53-deficient extra-thymic cancers. The data more than triple the literature on all non-mismatch repair deficient tumors for which mutations are identified by sequence analysis, allowing mutation frequency and pattern to be determined. Most tumors had a normal mutation frequency and a normal mutation pattern. Five tumors showed modest increases in mutation frequency (2.3-fold or less). Alterations in mutation patterns were uncommon, tumor-specific and not necessarily associated with increases in mutation frequency. Given the data from two spontaneous tumors (normal mutation frequency with an abnormal pattern in a p53-/- mouse and low mutation frequency in a p53+/+ control mouse), we hypothesize that tumors sometimes can carry a low mutation load. The study was not without certain caveats: mutation load could not be compared between tumor and normal tissue from the same animal; sample sizes for extra-thymic tumor types were small, and only point mutations and deletions, insertions and indels up to 2 kb were detected. However, the data clearly show key differences in tumors from p53+/- mice compared with mismatch repair deficient tumors; a lack of dramatic increase in mutation frequency and absence of a signature of mutation.

Animals↗
PubMed2004

The rad50 signature motif: essential to ATP binding and biological function.

The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies. One of the most critical of these is the Mre11/Rad50 (M/R) complex, which is present in all three biological kingdoms, but is not well-understood at the biochemical level. Previous structural analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases. Here, we present the crystallographic structure of the Rad50 mutant S793R. This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases. The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis. We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops. This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization. The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities. This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo. These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms.

Acid Anhydride Hydrolases↗
PubMed1994

Mutations in the K+ channel signature sequence.

Potassium channels share a highly conserved stretch of eight amino acids, a K+ channel signature sequence. The conserved sequence falls within the previously defined P-region of voltage-activated K+ channels. In this study we investigate the effect of mutations in the signature sequence of the Shaker channel on K+ selectivity determined under bi-ionic conditions. Nonconservative substitutions of two threonine residues and the tyrosine residue leave selectivity intact. In contrast, mutations at some positions render the channel nonselective among monovalent cations. These findings are consistent with a proposal that the signature sequence contributes to a selectivity filter. Furthermore, the results illustrate that the hydroxyl groups at the third and fourth positions, and the aromatic group at position seven, are not essential in determining K+ selectivity.

Amino Acid Sequence↗
PubMed2005

Mlh1-dependent suppression of specific mutations induced in vivo by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP).

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the potential impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We determined the effect of MMR status (Mlh1+/+ versus Mlh1-/-) on mutagenesis induced by the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) within cII and supFG1 transgene reporters. Despite being a lymphomagen in mice, PhIP was not mutagenic in thymus. In colon, PhIP exposure induced 3-fold more mutations in Mlh1-deficient mice compared to their Mlh1+/+ littermates. Similar induction was seen in Mlh1-/- small intestine. Analysis of mutational spectra revealed that G/C to T/A transversions, the "signature PhIP mutation", were induced to similar levels regardless of Mlh1 status. In contrast, Mlh1-/- mice exhibited hypermutability to frameshifts, G/C to A/T transitions, and G/C to C/G transversions. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs (PhIP-G/T, PhIP-G/G, and PhIP-G/loop mispairs). In contrast, the PhIP-G/A mispair is unlikely to be a MMR substrate. In addition, the similar induction of both transversions and transitions in Mlh1-/- mice suggests that mutagenic bypass of PhIP-G is similarly efficient with dATP, dTTP, and dGTP, in contrast to previously published conclusions. Our data suggests that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.

Adaptor Proteins, Signal Transducing↗