The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation.

Approximately 30% of human tumors examined for mutations in polymerase beta (pol beta) appear to express pol beta variant proteins (D. Starcevic, S. Dalal, and J. B. Sweasy, Cell Cycle 3:998-1001, 2004). Many of these variants result from a single amino acid substitution. We have previously shown that the K289M and I260M colon and prostate cancer variants, respectively, induce cellular transformation most likely due to sequence-specific mutator activity (S. Dalal et al., Biochemistry 44:15664-15673, 2005; T. Lang et al., Proc. Natl. Acad. Sci. USA 101:6074-6079, 2004; J. B. Sweasy et al., Proc. Natl. Acad. Sci. USA 102:14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol beta variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism.

The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient.

Approximately 30% of human tumors characterized to date express DNA polymerase beta (pol beta) variant proteins. Two of the polymerase beta cancer-associated variants are sequence-specific mutators, and one of them binds to DNA but has no polymerase activity. The Leu22Pro (L22P) DNA polymerase beta variant was identified in a gastric carcinoma. Leu22 resides within the 8 kDa amino terminal domain of DNA polymerase beta, which exhibits dRP lyase activity. This domain catalyzes the removal of deoxyribose phosphate during short patch base excision repair. We show that this cancer-associated variant has very little dRP lyase activity but retains its polymerase activity. Although residue 22 has no direct contact with the DNA, we report here that the L22P variant has reduced DNA-binding affinity. The L22P variant protein is deficient in base excision repair. Molecular dynamics calculations suggest that alteration of Leu22 to Pro changes the local packing, the loop connecting helices 1 and 2 and the overall juxtaposition of the helices within the N-terminal domain. This in turn affects the shape of the binding pocket that is required for efficient dRP lyase catalysis.

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.

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.

Circulating Cell-Free DNA to Determine the Fetal RHD Status in All Three Trimesters of Pregnancy.

OBJECTIVE: To estimate the accuracy of a new assay to determine the fetal RHD status using circulating cell-free DNA. METHODS: This was a prospective, observational study. Maternal blood samples were collected in each trimester of pregnancy in 520 nonalloimmunized RhD-negative patients. Plasma samples were analyzed for circulating cell-free DNA using the SensiGENE RHD test, which used primers for exons 4 and 7 as previously described and incorporated a new primer design for exon 5 of the RHD gene. Neonatal serology for RhD typing using cord blood at birth was undertaken and results were stored in a separate clinical database. After unblinding the data, results of the DNA analysis were compared with the neonatal serology. RESULTS: Inconclusive results secondary to the presence of the RHD pseudogene or an RHD variant were noted in 5.6%, 5.7%, and 6.1% of the first-, second-, and third-trimester samples, respectively. The incidence of false-positive rates for RhD (an RhD-negative fetus with an RHD-positive result) was 1.54% (95% confidence interval [CI] 0.42-5.44%), 1.53% (CI 0.42-5.40%), and 0.82% (CI 0.04-4.50%), respectively. There was only one false-negative diagnosis (an RhD-positive fetus with an RHD-negative result), which occurred in the first trimester (0.32%; 95% CI 0.08-1.78%). Genotyping for mismatches across repeated samples revealed that this error was related to mislabeling of samples from two patients collected on the same day at one of the collection sites. Overall test results were in agreement across all three trimesters (P>.99). CONCLUSION: Circulating cell-free DNA can accurately predict the fetal RhD status in all three trimesters of pregnancy.

New associations of the genetic polymorphisms in nicotinic receptor genes with the risk of lung cancer.

AIMS: Previous studies revealed association of lung cancer risk with single nucleotide polymorphisms (SNPs) in chromosome 15q25 region containing CHRNA5-CHRNA3-CHRNB4 nicotinic acetylcholine receptor (nAChR) subunit gene cluster. The genetic variations in other lung nAChRs remained unknown. In this study, we perform case-control analysis of CHRNA9 and CHRNA3 genes using 340 non-small cell lung cancer cases and 435 controls. MAIN METHODS: All exons, 3'UTR, intron 1 and parts of other introns surrounding exons 2-5 of CHRNA9 gene as well as exons 2, 3 of CHRNA3 gene and parts of surrounding intronic regions were sequenced. The study was controlled for gender, age and ethnicity related differences. Each SNP in analyzed groups was assessed by allele frequency, genotype distribution and haplotype analysis. KEY FINDINGS: The case-control analysis revealed that an increased risk is associated with two SNPs in CHRNA9, rs56159866 and rs6819385, and one in CHRNA3, rs8040868. The risk was reduced for three SNPs in CHRNA9, rs55998310, rs56291234, and newly discovered rs182073550, and also in carriers of the haplotype NP_060051.2 containing ancestral N442 variant of α9. SIGNIFICANCE: The nonsynonymous substitutions can produce receptors exhibiting unique ligand-binding and downstream signaling characteristics, synonymous as well all intronic SNPs may affect protein production at the transcriptional and/or translational levels, or just manifest association with cancer by genetic linkage to other alleles. Elucidation of the mechanisms by which individual genetic variations in α9 affect predisposition to lung cancer may lead to development of personalized approaches to cancer prevention and treatment as well as protection against tobacco consumption.

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.

Naturally occurring variants of human Α9 nicotinic receptor differentially affect bronchial cell proliferation and transformation.

Isolation of polyadenilated mRNA from human immortalized bronchial epithelial cell line BEP2D revealed the presence of multiple isoforms of RNA coded by the CHRNA9 gene for α9 nicotinic acetylcholine receptor (nAChR). BEP2D cells were homozygous for the rs10009228 polymorphism encoding for N442S amino acid substitution, and also contained mRNA coding for several truncated isoforms of α9 protein. To elucidate the biologic significance of the naturally occurring variants of α9 nAChR, we compared the biologic effects of overexpression of full-length α9 N442 and S442 proteins, and the truncated α9 variant occurring due to a loss of the exon 4 sequence that causes frame shift and early termination of the translation. These as well as control vector were overexpressed in the BEP2D cells that were used in the assays of proliferation rate, spontaneous vs. tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced cellular transformation, and tumorigenicity in cell culture and mice. Overexpression of the S442 variant significantly increased cellular proliferation, and spontaneous and NNK-induced transformation. The N442 variant significantly decreased cellular transformation, without affecting proliferation rate. Overexpression of the truncated α9 significantly decreased proliferation and suppressed cellular transformation. These results suggested that α9 nAChR plays important roles in regulation of bronchial cell growth by endogenous acetylcholine and exogenous nicotine, and susceptibility to NNK-induced carcinogenic transformation. The biologic activities of α9 nAChR may be regulated at the splicing level, and genetic polymorphisms in CHRNA9 affecting protein levels, amino acid sequence and RNA splicing may influence the risk for lung cancer.

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.

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.

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.