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PubMed1996

What drives codon choices in human genes?

Synonymous codon usage is based and the bias seems to be different in different organisms. Factors with proposed roles in causing codon bias include degree and timing of gene expression, codon-anticodon interactions, transcription and translation rate and fidelity, codon context, and global and local G + C content. We offer a new perspective and new methods for elucidating codon choices applied especially to the human genome. We present data supporting the thesis that codon choices for human genes are largely a consequence of two factors: (1) amino acid constraints, (2) maintaining DNA structures dependent on base-step conformational tendencies consistent with the organism's genome signature that is determined by genome-wide processes of DNA modification, replication and repair. The related codon signature defined as the dinucleotide relative abundances at the distinct codon positions (1,2), (2,3), and (3,4) (4 = 1 of the next codon) accommodates both the global genome signature and amino acid constraints. In human genes, codon positions (2,3) and (3,4) containing the silent site have similar codon signatures reflecting DNA symmetry. Strong CG and TA dinucleotide underrepresentation is observed at all codon positions as well as in non-coding regions. Estimates of synonymous codon usage based on codon signatures are in excellent agreement with the actual codon usage in human and general vertebrate genes. These properties are largely independent of the isochore compartment (G + C content), gene size, and transcriptional and translational constraints. We hypothesize that major influences on codon usage in human genes result from residue preferences and diresidue associations in proteins coupled to biases on the DNA level, related to replication and repair processes and/or DNA structural requirements.

Codon↗
PubMed1985

Fidelity of DNA polymerases isolated from regenerating liver chromatin of aging Mus musculus.

DNA polymerase-alpha and -beta were fractionated from the chromatin of regenerating liver of young and old mice. The DNA polymerases were resolved from each other and partially purified by DEAE-cellulose, phosphocellulose, and DNA-cellulose column chromatography. No significant age-related difference in the kinetics of heat inactivation was observed for either DNA polymerase. No age-dependent difference was found in the fidelity with which these enzymes copied phi X174 DNA. These results suggest that the functional properties of these DNA polymerases do not change with age as is postulated in some theories of aging.

Aging↗
PubMed2005

Polyphosphate kinase regulates error-prone replication by DNA polymerase IV in Escherichia coli.

The ppk gene encodes polyphosphate kinase (Ppk), an enzyme that catalyses the polymerization of inorganic phosphate into long chains of polyphosphate (polyP). An insertion mutation in ppk causes a decrease in adaptive mutation in Escherichia coli strain FC40. Adaptive mutation in FC40 mostly results from error-prone DNA polymerase IV (Pol IV), encoded by dinB; most of the antimutagenic phenotype of the ppk mutant disappears in a dinB mutant strain. In addition, the ppk mutant causes a decrease in growth-dependent mutations produced by overexpressing Pol IV. However, the amount of Pol IV protein is unchanged in the ppk mutant strain, indicating that the activity or fidelity of Pol IV is altered. Adaptive mutation is inhibited both by the absence of Ppk, which results in low amounts of polyP, and by overproduction of Ppk, which results in high amounts of polyP, suggesting that an optimal level of polyP is necessary. Taken together, these results suggest a novel mechanism involving polyP that directly or indirectly regulates DNA polymerase activity or fidelity.

Adaptation, Physiological↗
PubMed2004

In silico evidence for DNA polymerase-beta's substrate-induced conformational change.

Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.

Binding Sites↗
PubMed1983

Design of immobile nucleic acid junctions.

Nucleic acids that interact to generate structures in which three or more double helices emanate from a single point are said to form a junction. Such structures arise naturally as intermediates in DNA replication and recombination. It has been proposed that stable junctions can be created by synthesizing sets of oligonucleotides of defined sequence that can associate by maximizing Watson-Crick complementarity (Seeman N. C., 1981, Biomolecular Stereodynamics. Adenine Press, New York. 1: 269-278; Seeman, N. C., 1982, J. Theor. Biol. 99:237-247.) To make it possible to design molecules that will form junctions of specific architecture, we present here an efficient algorithm for generating nucleic acid sequences that optimize two fundamental properties: fidelity and stability. Fidelity refers to the relative probability of forming the junction complex relative to all alternative paired structures. Calculations are described that permit approximate prediction of the melting curves for junction complexes.

DNA↗
PubMed2003

Regulation of cell cycles is of key importance in human papillomavirus (HPV)-associated cervical carcinogenesis.

The rapid progress in molecular biology has allowed the identification of the genes involved in different functions of normal cells and has also improved our understanding of the mechanisms of human carcinogenesis. The human papillomavirus (HPV) is a small double-stranded DNA tumor virus and its genes can manipulate cell cycle control to promote viral persistence and replication. The E6 and E7 proteins of high-risk HPV bind to cell cycle regulatory proteins and interfere with both G1/S and G2/M cell cycle checkpoints much more effectively than the low-risk HPV. The difference between the ability of low and high-risk HPV types to induce immortalization and transformation may well lie in their abilities to interact with the various cell cycle components, resulting in the loss of multiple cell cycle checkpoints, which are important in host genome fidelity, thus potentially resulting in accumulation of genetic abnormalities. Cervical cancer is one of the leading malignancies in women worldwide, with substantial morbidity and mortality. According to current concepts, HPV is recognized as the single most important causal agent in the pathogenesis of this cancer. HPV infection clearly precedes the development of malignancy, while being regularly associated with cervical cancer precursor lesions (all grades of squamous intraepithelial lesions). HPV-infected low-grade squamous intraepithelial lesion (SIL) has three possible outcomes: a) it may regress; b) it can persist; or c) it can make a clinical progression to in situ or invasive carcinoma. It has been well established by prospective cohort studies that the spontaneous regression rate increases in parallel with follow-up duration. In contrast, the clinical progression of lesions usually takes place quite rapidly, i.e. during the first two years from diagnosis. The mechanisms responsible for this divergent clinical behavior of HPV-associated squamous intraepithelial lesions are largely unknown, but currently under intense study in different laboratories worldwide.

Cell Cycle↗
PubMed1996

RNA-catalysed RNA polymerization using nucleoside triphosphates.

The hypothesis that certain RNA molecules may be able to catalyse RNA replication is central to current theories of the early evolution of life. In support of this idea, we describe here an RNA that synthesizes RNA using the same reaction as that employed by protein enzymes that catalyse RNA polymerization. In the presence of the appropriate template RNA and nucleoside triphosphates, the ribozyme extends an RNA primer by successive addition of up to six mononucleotides. The added nucleotides are joined to the growing RNA chain by 3',5'-phosphodiester linkages. The ribozyme shows marked template fidelity: extension by nucleotides complementary to the template is up to 1,000 times more efficient than is extension by mismatched nucleotides.

Base Composition↗
PubMed1984

Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.

Chromatography, High Pressure Liquid↗
PubMed1995

Accuracy of Deoxynucleotide Incorporation by Soybean Chloroplast DNA Polymerases Is Independent of the Presence of a 3[prime] to 5[prime] Exonuclease.

DNA polymerase was purified from soybean (Glycine max) chloroplasts that were actively replicating DNA. The main form (form I) of the enzyme was associated with a low level of 3[prime] to 5[prime] exonuclease activity throughout purification, although the ratio of exonuclease to polymerase activity decreased with each successive purification step. A second form (form II) of DNA polymerase, which elutes from DEAE-cellulose at a higher salt concentration than form I, was devoid of any exonuclease activity. To assess the potential function of the 3[prime] to 5[prime] exonuclease in proofreading, the fidelity of deoxynucleotide incorporation was measured for form I DNA polymerase throughout purification. Despite the steadily decreasing ratio of 3[prime] to 5[prime] exonuclease to polymerase activity, the extent of misincorporation by form I enzyme remained unchanged during the final purification steps, suggesting that the exonuclease did not contribute to the accuracy of DNA synthesis by this polymerase. Fidelity of form I DNA polymerase, when compared with that of form II, revealed a higher level of misincorporation for form I enzyme, a finding that is consistent with the exonuclease playing little or no role in exonucleolytic proofreading.

Journal Article↗
PubMed1982

Genetic and physical map of a P1 miniplasmid.

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.

Base Sequence↗
PubMed1992

Concerted integration of viral DNA termini by purified avian myeloblastosis virus integrase.

Concerted integration of retroviral DNA termini, which produces a characteristic duplication of sequences at the integration site and formation of the proviral state, is a necessary step of the retroviral life cycle. We investigated the pairwise integration reaction catalyzed by purified avian retrovirus integrase by measuring the response to solution parameters and how the sequences of the viral termini, which comprise the avian imperfect inverted repeat, affect the reaction. When we optimized the reaction, an efficiency was achieved which approached that measured in systems using cytoplasmic extracts from virus-infected cells. The response of purified avian integrase to solution parameters was similar to that of the integration activity derived from cellular extracts. For strand transfer, the U3 viral terminal sequences were preferred to those of the U5 termini, a result we previously showed for the trimming reaction. That the sequence preference was the same for trimming and strand transfer may be further evidence that only one catalytic site is used for both reactions. A significant number of integration sites were sequenced. Interesting trends were found for the fidelity of the host duplications to the avian 6-bp duplication size, the clustering of the integration sites in the nonessential region of the lambda host DNA, and the sequence characteristics of the duplication sites.

Avian Myeloblastosis Virus↗
PubMed2005

Telomeres in evolution and evolution of telomeres.

This paper examines telomeres from an evolutionary perspective. In the monocot plant order Asparagales two evolutionary switch-points in telomere sequence are known. The first occurred when the Arabidopsis-type telomere was replaced by a telomere based on a repeat motif more typical of vertebrates. The replacement is associated with telomerase activity, but the telomerase has low fidelity and this may have implications for the binding of telomeric proteins. At the second evolutionary switch-point, the telomere and its mode of synthesis are replaced by an unknown mechanism. Elsewhere in plants (Sessia, Vestia, Cestrum) and in arthropods, the telomere "typical" of the group is lost. Probably many other groups with "unusual" telomeres will be found. We question whether telomerase is indeed the original end-maintenance system and point to other candidate processes involving t-loops, t-circles, rolling circle replication and recombination. Possible evolutionary outcomes arising from the loss of telomerase activity in alternative lengthening of telomere (ALT) systems are discussed. We propose that elongation of minisatellite repeats using recombination/replication processes initially substitutes for the loss of telomerase function. Then in more established ALT groups, subtelomeric satellite repeats may replace the telomeric minisatellite repeat whilst maintaining the recombination/replication mechanisms for telomere elongation. Thereafter a retrotransposition-based end-maintenance system may become established. The influence of changing sequence motifs on the properties of the telomere cap is discussed. The DNA and protein components of telomeres should be regarded--as with any other chromosome elements--as evolving and co-evolving over time and responding to changes in the genome and to environmental stresses. We describe how telomere dysfunction, resulting in end-to-end chromosome fusions, can have a profound effect on chromosome evolution and perhaps even speciation.

Chromosomes, Plant↗
PubMed1993

Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C-->T.A transversions in simian kidney cells.

A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells. The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter. Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector. Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site. This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA. This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E. coli. The predominant mutations observed (> 78%) were G-->T transversions targeted to the site of the lesion. The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E. coli. It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria.

Adamantane↗
PubMed2004

Quantitative signature for architectural organization of regulatory factors using intranuclear informatics.

Regulatory machinery for replication and gene expression is punctately organized in supramolecular complexes that are compartmentalized in nuclear microenvironments. Quantitative approaches are required to understand the assembly of regulatory machinery within the context of nuclear architecture and to provide a mechanistic link with biological control. We have developed 'intranuclear informatics' to quantify functionally relevant parameters of spatially organized nuclear domains. Using this informatics strategy we have characterized post-mitotic reestablishment of focal subnuclear organization of Runx (AML/Cbfa) transcription factors in progeny cells. By analyzing point mutations that abrogate fidelity of Runx intranuclear targeting, we establish molecular determinants for the spatial order of Runx domains. Our novel approach provides evidence that architectural organization of Runx factors may be fundamental to their tissue-specific regulatory function.

Animals↗
PubMed2004

The history of SIVS and AIDS: epidemiology, phylogeny and biology of isolates from naturally SIV infected non-human primates (NHP) in Africa.

Simian immunodeficiency virus (SIV) naturally infects non-human primates in Africa. To date, 40 SIVs have been described both in natural hosts and in heterologous species. These viruses are highly diverse and the majority cluster in 6 relatively equidistant phylogenetic lineages. At least 8 SIVs are currently considered as recombinant viruses, based on different clustering patterns in different genomic regions. Only three types of genomes are known, based on the number of accessory genes: vpr-containing genomes, vpr-vpx containing genomes and vpr-vpu-containing genomes. vpx resulted by a duplication of the vpr gene following non-homologous recombination and is characteristic of SIVs infecting the Papionini tribe of monkeys and HIV-2 in humans. vpu is characteristic of SIVcpz and HIV-1 and may have originated from a recombination involving SIVs from cercopitecini monkeys. SIV seems to be non-pathogenic in the vast majority of natural hosts in spite of a high levels of viral replication. This is probably a consequence of virus-host adaptation, in which the incubation period of the disease generally exceeds the life span of the African primate host. SIVs also have a high propensity for cross-species transmission. In the new host, the outcome may vary from inapparent infection to highly pathogenic, the former being reported for African monkeys, whereas the latter being observed in macaques and humans. The high diversity of SIVs was generated by a high mutation rate due to a low fidelity of the reverse-transcriptase and active viral and host cell turnover, host-dependent evolution and recombination. Cross-species transmission is not rare, however preferential host switching may drive the majority of cross-species transmissions. Numerous SIVs tested so far are able to grow in vitro on human PBMC, therefore it has been postulated that SIV represents a threat for infection of humans in Central Africa and that AIDS is a zoonosis. However, although the simian origin of the two HIV types is broadly acknowledged, there are no data that AIDS is acquired like a zoonosis. SIV may undergo adaptation in the new human host in order to emerge in the general population. The study of SIV in their natural hosts should provide important clues to the real threat to human populations and also elucidate the mechanisms associated with a long-term persistent viral infection without clinical consequences for the host.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Resolving a fidelity paradox: why Escherichia coli DNA polymerase II makes more base substitution errors in AT- compared with GC-rich DNA.

The activity of DNA polymerase-associated proofreading 3'-exonucleases is generally enhanced in less stable DNA regions leading to a reduction in base substitution error frequencies in AT- versus GC-rich sequences. Unexpectedly, however, the opposite result was found for Escherichia coli DNA polymerase II (pol II). Nucleotide misincorporation frequencies for pol II were found to be 3-5-fold higher in AT- compared with GC-rich DNA, both in the presence and absence of polymerase processivity subunits, beta dimer and gamma complex. In contrast, E. coli pol III holoenzyme, behaving "as expected," exhibited 3-5-fold lower misincorporation frequencies in AT-rich DNA. A reduction in fidelity in AT-rich regions occurred for pol II despite having an associated 3'-exonuclease proofreading activity that preferentially degrades AT-rich compared with GC-rich DNA primer-template in the absence of DNA synthesis. Concomitant with a reduction in fidelity, pol II polymerization efficiencies were 2-6-fold higher in AT-rich DNA, depending on sequence context. Pol II paradoxical fidelity behavior can be accounted for by the enzyme's preference for forward polymerization in AT-rich sequences. The more efficient polymerization suppresses proofreading thereby causing a significant increase in base substitution error rates in AT-rich regions.

Base Sequence↗
PubMed2000

Cancer risk and the ATM gene: a continuing debate.

Deficiencies in the ability of cells to sense and repair damage in individuals with rare genetic instability syndromes increase the risk of developing cancer. Ataxia-telangiectasia (A-T), such a condition, is associated with a high incidence of leukemia and lymphoma that develop in childhood. Although A-T is an autosomal recessive disorder, some penetrance appears in individuals with one mutated ATM gene (A-T carriers), namely, an increased risk of developing breast cancer. The gene mutated in A-T, designated ATM, is homologous to several DNA damage recognition and cell cycle checkpoint control genes from other organisms. Recent studies suggest that ATM is activated primarily in response to double-strand breaks, the major cytotoxic lesion caused by ionizing radiation, and can directly bind to and phosphorylate c-Abl, p53, and replication protein A (RPA). Analysis of ATM mutations in patients with A-T or with sporadic non-A-T cancers has suggested the existence of two classes of ATM mutation: null mutations leading to A-T and dominant negative missense mutations predisposing to cancer in the heterozygous state. Studies with A-T mouse models have helped determine the basis of lymphoid tumorigenesis in A-T and have shown that ATM plays a critical role in maintaining genetic stability by ensuring high-fidelity execution of chromosomal events. Thus, ATM appears to act as a caretaker of the genome.

Ataxia Telangiectasia↗
PubMed1995

Frontier questions about sister chromatid separation in anaphase.

Sister chromatid separation in anaphase is an important event in the cell's transmission of genetic information to a descendent. It has been investigated from different aspects: cell cycle regulation, spindle and chromosome dynamics within the three-dimensional cell architecture, transmission fidelity control and cellular signaling. Integrated studies directed toward unified understanding are possible using multidisciplinary methods with model organisms. Ubiquitin-dependent proteolysis, protein dephosphorylation, an unknown function by the TPR repeat proteins, chromosome transport by microtubule-based motors and DNA topological change by DNA topoisomerase II are all necessary for progression from metaphase to anaphase. Chromosome condensation, mitotic kinetochore function and spindle formation require a larger number of proteins, which are prerequisites for successful sister chromatid separation. Factors that help to retain sister chromatid connection after replication and prevent premature separation remain to be determined. Although sister chromatid separation occurs in anaphase, gene functions in other cell cycle stages also ensure the progression of correct chromatid separation.

Anaphase↗