Flexible tethering of primase and DNA Pol α in the eukaryotic primosome

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA–DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA–DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.     

The 3′ → 5′ exonucleases of both DNA polymerases δ and ε participate in correcting errors of DNA replication in Saccharomyces cerevisiae

DNA polymerases II (ε) and III(δ) are the only nuclear DNA polymerases known to possess an intrinsic 3′ → 5′ exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase δ and ε 3′ → 5′ exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10² and 10¹, respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 × 10³-fold. Analysis of mutation rates of double mutants indicated that the 3′ → 5′ exonucleases of DNA polymerases δ and ε can act competitively and that, like the 3′ → 5′ exonuclease of DNA polymerase δ the 3′ → 5′ exonuclease of DNA polymerase ε acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3′ → 5′ exonucleases of DNA polymerases δ and ε act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.     

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal β-glycosidases

Sβgly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of Sβgly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure–function relationships in these enzymes. Received: May 20, 1999 / Accepted: January 10, 2000     

Intrinsic properties of the two replicative DNA polymerases of Pyrococcus abyssi in replicating abasic sites: possible role in DNA damage tolerance?

Spontaneous and induced abasic sites in hyperthermophiles DNA have long been suspected to occur at high frequency. Here, Pyrococcus abyssi was used as an attractive model to analyse the impact of such lesions onto the maintenance of genome integrity. We demonstrated that endogenous AP sites persist at a slightly higher level in P. abyssi genome compared with Escherichia coli. Then, the two replicative DNA polymerases, PabpolB and PabpolD, were characterized in presence of DNA containing abasic sites. Both Pabpols had abortive DNA synthesis upon encountering AP sites. Under running start conditions, PabpolB could incorporate in front of the damage and even replicate to the full-length oligonucleotides containing a specific AP site, but only when present at a molar excess. Conversely, bypassing activity of PabpolD was strictly inhibited. The tight regulation of nucleotide incorporation opposite the AP site was assigned to the efficiency of the proof-reading function, because exonuclease-deficient enzymes exhibited effective TLS. Steady-state kinetics reinforced that Pabpols are high-fidelity DNA polymerases onto undamaged DNA. Moreover, Pabpols preferentially inserted dAMP opposite an AP site, albeit inefficiently. While the template sequence of the oligonucleotides did not influence the nucleotide insertion, the DNA topology could impact on the progression of Pabpols. Our results are interpreted in terms of DNA damage tolerance.     

Ancient DNA enables timing of the pleistocene origin and holocene expansion of two adélie penguin lineages in antarctica

The timing of divergent events in history is one of the central goals of contemporary evolutionary biology. Such studies are however dependent on accurate evolutionary rates. Recent developments in ancient DNA analysis enable the estimation of more accurate evolutionary rates and therefore more accurate timing of divergence events. Consequently, this leads to a better understanding of changes in populations through time. We use an evolutionary rate calculated from ancient DNA of Adélie penguins (Pygoscelis adeliae) to time divergent events in their history. We report the presence of two distinct and highly variable mitochondrial DNA lineages and track changes in these lineages through space and time. When the ancient DNA and the phylogenetic rates are used to estimate the time of origin of the lineages, two very different estimates resulted. In addition, these same rates provide very different estimates of the time of expansion of these lineages. We suggest that the rate calculated from ancient DNA is more consistent with the glacial history of Antarctica and requires fewer assumptions than does a narrative based on the phylogenetic rate. Finally, we suggest that our study indicates an important new role for ancient DNA studies in the timing of divergent events in history.     

Bowman–Birk inhibitors in Lens: identification and characterization of two paralogous gene classes in cultivated lentil and wild relatives

In order to investigate the genetic structure of lentil Bowman–Birk inhibitors (BBIs), primers were designed on pea BBI sequences. The sequences obtained from lentil DNA, using these primers, indicate that lentil possesses at least two paralogous genes. Protein sequences translated in silico from lentil DNA sequences suggest that the two coded proteins are highly similar to Pisum trypsin inhibitor TI1 and TI6 BBIs, respectively. In fact, both are double-headed inhibitors, one class showing the presence of a trypsin- and a chymotrypsin-reactive site, the other showing two trypsin-inhibition sites, similar to pea TI1 and TI6, respectively. The same primers were used to amplify sequences from the DNA of other Lens species. The results strongly support that all species of Lens possess the same classes of BBI coding genes, orthologous to those identified in the cultivated lentil. Lens nigricans showed the most diverging sequences both at the nucleotide and the amino acid level. The similarity of the two gene classes identified in the genus Lens to those of Pisum and the observations that the patterns of expression of the Lens genes are equivalent to those of pea orthologous genes, possibly imply that BBIs in Lens are coded by gene classes with similar genome organization and function to those of pea. Finally, a phyletic analysis, based on the comparison of sequences obtained from other species belonging to the Vicieae tribe of the Fabaceae family, strongly suggests that all Vicieae could have a similar genome organization and function for BBI genes, and that this could be a general rule in all the Fabaceae family.Publication of the Institute of Plant Genetics N. 50     

Analyses of a multi-parent population derived from two diverse alfalfa germplasms: testcross evaluations and phenotype–DNA associations

In a previous study, we showed that the genetic variation present in the Medicago sativa subsp. sativa Peruvian and M. sativa subsp. falcata WISFAL germplasms could be used to improve forage yields when favorable alleles were recombined and used in hybrid combination with cultivated alfalfa. In this paper, we present testcross forage yield and fall growth data for two seasons of a C0 population generated after intermating the Peruvian × WISFAL population for several generations. In addition, we conducted marker-trait association analysis as an attempt to identify Peruvian and WISFAL genomics regions affecting the targeted traits. Five and seven genomic regions were found significantly associated with forage yield and fall growth, respectively. In the case of fall growth, alleles from both accessions were positively associated with plant height. However, more alleles from WISFAL were positively associated with forage yield than from Peruvian. WISFAL is known for its winter hardiness and genomic regions with large effects on winter survival may have masked the effect of forage yield from Peruvian. The fact that most of the genomic regions discovered in this study have been previously associated with traits involved in winter hardiness validates our findings and suggests that associations between DNA fragments and agronomic traits can be detected without the necessity of developing bi-parental mapping populations.     

Evolution of viruses and cells: do we need a fourth domain of life to explain the origin of eukaryotes?

The recent discovery of diverse very large viruses, such as the mimivirus, has fostered a profusion of hypotheses positing that these viruses define a new domain of life together with the three cellular ones (Archaea, Bacteria and Eucarya). It has also been speculated that they have played a key role in the origin of eukaryotes as donors of important genes or even as the structures at the origin of the nucleus. Thanks to the increasing availability of genome sequences for these giant viruses, those hypotheses are amenable to testing via comparative genomic and phylogenetic analyses. This task is made very difficult by the high evolutionary rate of viruses, which induces phylogenetic artefacts, such as long branch attraction, when inadequate methods are applied. It can be demonstrated that phylogenetic trees supporting viruses as a fourth domain of life are artefactual. In most cases, the presence of homologues of cellular genes in viruses is best explained by recurrent horizontal gene transfer from cellular hosts to their infecting viruses and not the opposite. Today, there is no solid evidence for the existence of a viral domain of life or for a significant implication of viruses in the origin of the cellular domains.     

Decision-making during NHEJ: a network of interactions in human Polμ implicated in substrate recognition and end-bridging

Human Polμ is a DNA polymerase belonging to the X family that has been implicated in the non-homologous end-joining (NHEJ) pathway during repair of double-strand breaks in DNA. Loop1 is a flexible piece of Polμ which has a critical role during terminal transferase and end-joining activities: it acts as a pseudo-template when the template strand is discontinuous or unavailable, whilst diffusing away if present to avoid steric clashes. Mutational analysis and inspection of the 3D structures available allowed us to identify a network of residues in charge of sensing the presence or absence of discontinuities in the template strand, which will in turn determine the final position adopted by Loop1. This network is formed by the previously uncharacterized thumb mini-loop (NSH motif) and the positively charged helix N, which contribute to the correct positioning of Loop1 and to juxtapose the discontinuous template strand during NHEJ of incompatible ends. Accordingly, single mutation of specific conserved residues in these motifs, whilst irrelevant in most of the cases for gap filling, largely affected terminal transferase and end-joining activities. Other point mutations in the ‘hinges’ of Loop1, such as residues Phe³⁸⁵ or Phe³⁸⁹, corroborated the flexibility requirements of this motif.     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

Molecular Evolution of Daphnia Immunity Genes: Polymorphism in a Gram-Negative Binding Protein Gene and an α-2-Macroglobulin Gene

Studies of DNA polymorphism have shown that some immune system genes of mammals and plants are exceptionally diverse, indicating that coevolution between these taxa and their parasites mediates positive selective sweeps and/or balancing selection. The genes of the arthropod immune system remain comparatively unstudied. We isolated two putative immune system genes from the cladoceran crustacean Daphnia and examined DNA sequence diversity. For one gene, encoding a putative gram-negative binding protein, we found evidence of only purifying selection, indicating that this gene is under strong functional constraint and that selection acts to eliminate amino acid variation. For another gene, encoding a putative -2-macroglobulin, we found evidence of positive selection, indicating the possible involvement of this gene in a host–parasite arms race. We discuss the assumed function of these genes and offer speculation regarding which components of the arthropod immune system might experience diversifying adaptive evolution.[Reviewing Editor: Dr. Martin Kreitman]     

Levels of 5-hydroxymethyl-2′-deoxyuridine in DNA from women participating in an intervention trial of low-fat and low-energy diets

Oxidative DNA damage in blood appears to be useful as a marker of systemic oxidative stress levels. Dietary factors such as fat and energy intakes have been indicated to affect oxidative stress levels, and this may be an important mechanism by which diet can modulate cancer risk. The primary objective of this study was to investigate the effects of dietary intervention in premenopausal women on the levels of one type of oxidative DNA damage: 5-hydroxymethyl-2′-deoxyuridine. The trial randomly assigned women to control, low-fat, low-energy or combination low-fat/low-energy diets for 12?weeks. Blood samples were obtained every 2?weeks, and DNA was analysed for the levels of 5-hydroxymethyl-2′-deoxyuridine. Levels of DNA damage declined with time in each diet arm, including the control arm. The decreases were greater in the two arms with low-energy intake, but not significantly so. The numbers of women who exhibited decreased 5-hydroxymethyl-2′-deoxyuridine levels at 12?weeks versus baseline levels, however, was significantly greater in women assigned to any intervention diet (79%) than in the control arm (50%). Low-fat and low-energy diets therefore had a small effect on changes in oxidative DNA damage levels. The women participating in this study were not selected on the basis of increased cancer risk; therefore, they may have had low baseline levels of damage that were not amenable to further reduction by dietary change.     

Bergmann and converse bergmann latitudinal clines in arthropods: two ends of a continuum?

Two seemingly opposite evolutionary patterns of clinal variationin body size and associated life history traits exist in nature.According to Bergmann's rule, body size increases with latitude,a temperature effect. According to the converse Bergmann rule,body size decreases with latitude, a season length effect. Athird pattern causally related to the latter is countergradientvariation, whereby populations of a given species compensateseasonal limitations at higher latitudes by evolving fastergrowth and larger body sizes compared to their low latitudeconspecifics. We discuss these patterns and argue that theyare not mutually exclusive because they are driven by differentenvironmental causes and proximate mechanisms; they thereforecan act in conjunction, resulting in any intermediate pattern.Alternatively, Bergmann and converse Bergmann clines can beinterpreted as over- and undercompensating countergradient variation,respectively. We illustrate this with data for the wide-spreadyellow dung fly, Scathophaga stercoraria (Diptera: Scathophagidae),which in Europe shows a Bergmann cline for size and a converseBergmann cline (i.e., countergradient variation) for developmenttime. A literature review of the available evidence on arthropodlatitudinal clines further shows a patterned continuum of responses.Converse Bergmann clines due to end-of-season time limitationsare more common in larger species with longer development times.Our study thus provides a synthesis to the controversy aboutthe importance of Bergmann's rule and the converse Bergmannrule in nature.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3'----5')-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable 'module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.