A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome

The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.     

Is gene deletion in eukaryotes sequence-dependent? A study of nine deletion junctions and nineteen other deletion breakpoints in intron 7 of the human dystrophin gene

Although large deletions comprise 65% of the mutations that underlie most cases of Duchenne and Becker muscular dystrophies, the DNA sequence characteristics of the deletions and the molecular processes leading to their formation are largely unknown. Intron 7 of the human dystrophin gene is unusually large (110 kb) and a substantial number of deletions have been identified with endpoints within this intron. The distribution of 28 deletion endpoints was mapped to local sequence elements by PCR. The break points were distributed among unique sequence, LINE-1, Alu, MIR, MER and microsatellite sequences with frequencies expected from the frequency of those sequences in the intron. Thus, deletions in this intron are not associated primarily with any one of those sequences in the intron. Nine deletion junctions were amplified and sequenced. Eight were deletions between DNA sequences with minimal homology (0–4 bp) and are therefore unlikely to be products of homologous recombination. In the ninth case, a complex rearrangement was found to be consistent with unequal recombinational exchange between two Alu sequences coupled with a duplication. We have hypothesized that a paucity of matrix attachment regions in this very large intron expanded by the insertion of many mobile elements might provoke a chromatin structure that stimulates deletions (McNaughton et al., 1997, Genomics 40, 294–304). The data presented here are consistent with that idea and demonstrate that the deletion sequences are not usually produced by homologous DNA misalignments.     

Identification of a mutation within the structural gene for the α subunit of DNA-dependent RNA polymerase of E. Coli

Summary An E. coli mutant rpoA109 unable to support the growth of phage P2 produces DNA-dependent RNA polymerase with an altered subunit. Histidine is substituted for leucine in one tryptic peptide from the mutant subunit. The existence of only one rpoA gene within the E. coli chromosome is indicated.     

Evolution of “determinants” in sex-determination: A novel hypothesis for the origin of environmental contingencies in avian sex-bias

Sex-determination is commonly categorized as either “genetic” or “environmental”—a classification that obscures the origin of this dichotomy and the evolution of sex-determining factors. The current focus on static outcomes of sex-determination provides little insight into the dynamic developmental processes by which some mechanisms acquire the role of sex determinants. Systems that combine “genetic” pathways of sex-determination (i.e., sex chromosomes) with “environmental” pathways (e.g., epigenetically induced segregation distortion) provide an opportunity to examine the evolutionary relationships between the two classes of processes and, ultimately, illuminate the evolution of sex-determining systems. Taxa with sex chromosomes typically undergo an evolutionary reduction in size of one of the sex chromosomes due to suppressed recombination, resulting in pronounced dimorphism of the sex chromosomes, and setting the stage for emergence of epigenetic compensatory mechanisms regulating meiotic segregation of heteromorphic sex chromosomes. Here we propose that these dispersed and redundant regulatory mechanisms enable environmental contingency in genetic sex-determination in birds and account for frequently documented context-dependence in avian sex-determination. We examine the evolution of directionality in such sex-determination as a result of exposure of epigenetic regulators of meiosis to natural selection and identify a central role of hormones in integrating female reproductive homeostasis, resource allocation to oocytes, and offspring sex. This approach clarifies the evolutionary relationship between sex-specific molecular genetic mechanisms of sex-determination and non-sex-specific epigenetic regulators of meiosis and demonstrates that both can determine sex. Our perspective shows how non-sex-specific mechanisms can acquire sex-determining function and, by establishing the explicit link between physiological integration of oogenesis and sex-determination, opens new avenues to the studies of adaptive sex-bias and sex-specific resource allocation in species with genetic sex-determination.     

Ribonucleotide reductases: the link between an RNA and a DNA world?

The structures of a class III ribonucleotide reductase (RNR) and pyruvate formate lyase exhibit striking homology within their active site domains with respect to each other and to the previously published structure of a class I RNR. The common structures and the common complex-radical-based chemistry of these systems, as well as of the class II RNRs, suggest that RNRs evolved by divergent evolution and provide an essential link between the RNA and DNA world.     

Induction of alkaline phosphatase and the glycoprotein hormone α subunit in HeLa cells by inhibitors of DNA polymerase

Levels of the glycoprotein hormone α subunit and alkaline phosphatase activity were increased in cultures of HeLa S₃ cells exposed to aphidicolin (0.2–10 μg/ml) or phosphonoformic acid (0.1–3 mm), inhibitors of DNA polymerase α. Induction was dependent on both the concentration and duration of exposure to the inhibitors and was prevented by cycloheximide and actinomycin D. Limited characterization of the induced α subunit and alkaline phosphatase activity suggest that they are similar to the uninduced proteins expressed by this cell line. Induction of both proteins by aphidicolin and phosphonoformic acid was enhanced by the simultaneous addition of 3 mm sodium butyrate but was depressed by 1 mm hydroxy urea. In contrast, both butyrate and hydroxy urea cause induction of these proteins when added alone to HeLa cultures. It is unlikely that a direct relationship exists between protein induction and the inhibition of DNA synthesis produced by aphidicolin and phosphonoformic acid since the concentrations required to produce half-maximal induction are 5 to 10 times greater than those needed to inhibit replication by 50%.     

Chlorella chloroplast DNA sequence containing a gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a part of a possible gene for the β′ subunit of RNA polymerase

The sequence of a 2782 bp fragment of the chloroplast genome of Chlorella ellipsoidea has been determined. The region includes the entire gene (rbcL) for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase and a sequence (rpoC-like) similar to part of the gene for the subunit of E. coli RNA polymerase which is oriented in same direction as rbcL. The arrangement is rpoC-like — 446 bp — rbcL. The rbcL gene codes for a polypeptide of 475 amino acids whose sequence shows 88% homology with those of tobacco and spinach, 94% homology with that of Chlamydomonas, and 85% homology with that of Anacystis. The putative rbcL promoter sequence has homology with E. coli promoter sequences and its putative terminator sequence is capable of forming a stem-and-loop structure.     

SULT1A1 gene deletion in BRCA2-associated male breast cancer: a link between genes and environmental exposures?

SULT1A1, a member of sulfotransferase superfamily, is a drug and hormone metabolizing enzyme involved in the metabolism of a variety of potential mammary carcinogens of endogenous and exogenous origin. Interestingly, the metabolic activity of SULT1A1 can be affected by variations in gene copy number. Male Breast Cancer (MBC) is a rare disease and less investigated disease compared to female BC (FBC). As in FBC, the concurrent effects of genetic risk factors, particularly BRCA2 mutations, increased exposure to estrogens and environmental carcinogens play a relevant role in MBC. By quantitative real‐time PCR with TaqMan probes, we investigated the presence of SULT1A1 gene copy number variations (CNVs) in a series of 72 MBCs. SULT1A1 gene deletion was observed in 10 of the 72 MBCs (13.9%). In a multivariate analysis association between BRCA2 mutation and SULT1A1 gene deletion emerged (p = 0.0005). Based on the evidence that the level of SULT1A1 enzyme activity is correlated with CNV, our data suggest that in male breast tumors SULT1A1 activity may be decreased. Thus, it can be hypothesized that in a proportion of MBCs, particularly in BRCA2‐associated MBCs, the level of estrogens and environmental carcinogens exposure might be increased suggesting a link between gene and environmental exposure in the pathogenesis of MBC.     

A deletion in the human QP-C gene causes a complex III deficiency resulting in hypoglycaemia and lactic acidosis

Mitochondrial respiratory chain complex III (ubiquinol-cytochrome c reductase) consists of 11 subunits, only one (cytochrome b) being encoded by the mitochondrial DNA. Disorders of complex III are comparatively rare but are nevertheless present as a clinically heterogeneous group of diseases. To date, no mutation in any of the nuclear-encoded subunits has been described. We report here a deletion in the nuclear gene UQCRB encoding the human ubiquinone-binding protein of complex III (QP-C subunit or subunit VII) in a consanguineous family with an isolated complex III defect. In the proband, a homozygous 4-bp deletion was identified at nucleotides 338-341 of the cDNA predicting both a change in the last seven amino acids and an addition of a stretch of 14 amino acids at the C-terminal end of the protein. Both parents were found to be heterozygous for the deletion, which was absent from 55 controls. Low temperature (-196 degrees C) spectral studies performed on isolated mitochondria from cultured skin fibroblast of the proband showed a decreased cytochrome b content suggestive of a role for the QP-C subunit in the assembly or maintenance of complex III structure.     

Coding in the Noncoding DNA Strand: A Novel Mechanism of Gene Evolution?

The question whether the noncoding DNA strand had or still has the capability for encoding functional polypeptides has been addressed in several articles. The theoretical background of the views advocating this idea arose from two groups of findings. One of them was based on various observations implying that the genetic code was adapted for double-strand coding. The other group of theories arose from the observation of gene-length overlapping open reading frames (O-ORFs) on the antisense DNA strand in a number of genes. In fact, the above theories, which I term selectionist, conceive a novel conception of gene evolution, proposing that new genes can be created by the utilization of antisense DNA strand. In contrast, neutralist theory claims that the O-ORFs are mere by-products of evolutionary processes acting to create special codon usage and base distribution patterns in the coding sequences. Received: 16 June 2000 / Accepted: 31 August 2000     

Intranuclear dynamics of DNA polymerase α differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase α activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase α activity, with the majority (60–75%) localized in soluble nucleoplasm and a significant amount (13–20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase α was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60–80%) of DNA polymerase α activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60–80%) of DNA polymerase β activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase α molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase α which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase β in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.