Association of single nucleotide polymorphisms of nicotinic acetylcholine receptor subunits with cervical neoplasia.

AIMS: Cholinergic signaling, particularly in response to non-physiological ligands like nicotine, stimulates carcinogenesis of a variety of tissue types including epithelia of the cervix uteri. Cholinergic signaling is mediated by nicotinic acetylcholine receptors (nAChRs), which are pentamers formed by subsets of 16 nAChR subunits. Recent literature suggests that single nucleotide polymorphisms (SNPs) of some of these subunits, notably alpha5, are risk factors for developing lung cancer in smokers as well as in non-smokers. MAIN METHODS: We have studied the prevalence of four SNPs in the alpha5, alpha9, and beta1 subunits, which are expressed in cervical cells, in 456 patients with cervical cancers, precursor lesions, and healthy controls from two cohorts in Mexico. KEY FINDINGS: A SNP in the alpha9 subunit, the G allele of rs10009228 (alpha9, A>G) shows a significant trend in the combined cohort, indicating that this allele constitutes a risk factor for neoplastic progression. The A allele of the SNP rs16969968 (alpha5, G>A), which correlates with the development of lung cancer, shows a non-significant trend to be associated with cervical lesions. Two other SNPs, rs55633891 (alpha9, C>T) and rs17856697 (beta1, A>G), did not exhibit a significant trend. SIGNIFICANCE: Our study points to a potential risk factor of cervical carcinogenesis with importance for DNA diagnosis and as a target for intervention.

The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III theta subunit, is expressed during both lysogenic and lytic growth stages.

The bacteriophage P1 hot gene product is a homolog of the theta subunit of E. coli DNA polymerase III. Previous studies with hot cloned on a plasmid have shown that Hot protein can substitute for theta, as evidenced by its stabilizing effect on certain dnaQ mutator mutants carrying an unstable pol III proofreading subunit (epsilon subunit). These results are consistent with Hot, like theta, being a replication protein involved in stabilizing the intrinsically unstable epsilon proofreading function. However, the function of hot for the viral life cycle is less clear. In the present study, we show that the hot gene is not essential. Based on its promoter structure, hot has been previously classified as a "late" phage gene, a property that is not easily reconciled with a presumed replication function. Here, we clarify this issue by demonstrating that P1 hot is actively expressed both during the lysogenic state and in the early stages of a lytic induction, in addition to its expression in the late stage of phage development. The results indicate that P1 hot has a complex expression pattern, compatible with a model in which Hot may affect the host replication machinery to benefit overall phage replication.

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex.

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.

Mutator and antimutator effects of the bacteriophage P1 hot gene product.

The Hot (homolog of theta) protein of bacteriophage P1 can substitute for the Escherichia coli DNA polymerase III theta subunit, as evidenced by its stabilizing effect on certain dnaQ mutants that carry an unstable polymerase III epsilon proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of various E. coli strains (mutator effect). The hot mutator effect differs from the one caused by the lack of theta. Experiments using chimeric theta/Hot proteins containing various domains of Hot and theta along with a series of point mutants show that both N- and C-terminal parts of each protein are important for stabilizing the epsilon subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.

The bacteriophage P1 hot gene product can substitute for the Escherichia coli DNA polymerase III {theta} subunit.

The theta subunit (holE gene product) of Escherichia coli DNA polymerase (Pol) III holoenzyme is a tightly bound component of the polymerase core. Within the core (alpha-epsilon-theta), the alpha and epsilon subunits carry the DNA polymerase and 3' proofreading functions, respectively, while the precise function of theta is unclear. holE homologs are present in genomes of other enterobacteriae, suggestive of a conserved function. Putative homologs have also been found in the genomes of bacteriophage P1 and of certain conjugative plasmids. The presence of these homologs is of interest, because these genomes are fully dependent on the host replication machinery and contribute few, if any, replication factors themselves. To study the role of these theta homologs, we have constructed an E. coli strain in which holE is replaced by the P1 homolog, hot. We show that hot is capable of substituting for holE when it is assayed for its antimutagenic action on the proofreading-impaired dnaQ49 mutator, which carries a temperature-sensitive epsilon subunit. The ability of hot to substitute for holE was also observed with other, although not all, dnaQ mutator alleles tested. The data suggest that the P1 hot gene product can substitute for the theta subunit and is likely incorporated in the Pol III complex. We also show that overexpression of either theta or Hot further suppresses the dnaQ49 mutator phenotype. This suggests that the complexing of dnaQ49-epsilon with theta is rate limiting for its ability to proofread DNA replication errors. The possible role of hot for bacteriophage P1 is discussed.

Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III.

DNA polymerase III, the main replicative polymerase of E. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. It was recently discovered that E. coli bacteriophage P1 encodes a theta homolog, named HOT. The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR. The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different. Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT. As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein.