[Is health really the most important value? - Results of a representative survey of the German general population concerning the subjective meaning of health]. / Ist Gesundheit das höchste Gut? - Ergebnisse einer bevölkerungsrepräsentativen Umfrage zur subjektiven Bedeutung von Gesundheit.

INTRODUCTION: The aim of this study was to investigate which meaning is attributed to health by the general population. Furthermore, the relationship between health satisfaction and health importance was also analysed. METHOD: A sample of 4,808 representatively selected subjects from the general German population judged the importance and the satisfaction with several life domains, including health, using the questions on life satisfaction FLZ (M). Moreover, sociodemographic variables (sex, age, socioeconomic status) and psychological variables (self-esteem, resilience, anxiety and depression) were collected. RESULTS: Health is the most important life domain. The importance of health increases with increasing age. However, there are no sex differences and SES (socio-economic status) differences concerning the importance of health. Subjective satisfaction with health and health importance are only marginally correlated (r=0.08). High degrees of self-esteem and resilience are associated with a high importance of health. Anxiety and depression show only weak relationships to the importance of health. CONCLUSIONS: In the German general population health has a very high subjective significance. This is not only true for handicapped or ill people, but for all subsamples of the society. Therefore, a general plea for an understanding of the importance of health is not necessary, not even for subgroups. Preventive activity can be based on the general understanding of the meaning of health, but it should pursue specific health- related goals for target groups.

DNA polymerases and oxidative damage: friends or foes?

DNA is modified by many mutagens, including reactive oxygen species (ROS). When ROS react with DNA, various kinds of modified base and/or sugar moieties are produced. One of the most important oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). Contrary to normal deoxyguanosine, 8-oxo-G favors a syn conformation, enabling it to form a Hoogsteen base pair with adenine which resembles a normal Watson-Crick base pair in shape and geometry. As a consequence, most human DNA polymerases (pols) studied so far show significant error-prone bypass of 8-oxo-G. The 1,2-dihydro-2-oxoadenine (2-OH-A) is another common DNA lesion produced by ROS. 2-OH-A possesses significant mutagenic potential in living cells. When challenged with a 2-OH-A lesion on the template, DNA pols often misinsert G and C nucleotides, with various efficiencies depending upon the sequence context. We have recently shown that human DNA pol lambda is extremely efficient in performing error-free bypass of both 8-oxo-G and 2-OH-A lesions, and that its efficiency is positively modulated by the auxiliary factors proliferating cell nuclear antigen and replication protein A. In this review we will summarize the most recent advancements in the field of oxidative DNA damage tolerance with special emphasis on the pro- and anti-mutagenic roles of DNA pols and auxiliary proteins.

Modulation of cellular response to cisplatin by a novel inhibitor of DNA polymerase beta.

DNA polymerase beta (Pol beta) is an error-prone enzyme whose up-regulation has been shown to be a genetic instability enhancer as well as a contributor to cisplatin resistance in tumor cells. In this work, we describe the isolation of new Pol beta inhibitors after high throughput screening of 8448 semipurified natural extracts. In vitro, the selected molecules affect specifically Pol beta-mediated DNA synthesis compared with replicative extracts from cell nuclei. One of them, masticadienonic acid (MA), is particularly attractive because it perturbs neither the activity of the purified replicative Pol delta nor that of nuclear HeLa cell extracts. With an IC50 value of 8 microM, MA is the most potent of the Pol beta inhibitors found so far. Docking simulation revealed that this molecule could substitute for single-strand DNA in the binding site of Pol beta by binding Lys35, Lys68, and Lys60, which are the main residues involved in the interaction Pol beta/single-strand DNA. Selected inhibitors also affect the Pol beta-mediated translesion synthesis (TLS) across cisplatin adducts; MA was still the most efficient. Therefore, masticadienonic acid sensitized the cisplatin-resistant 2008C13*5.25 human tumor cells. Our data suggest that molecules such as masticadienonic acid could be suitable in conjunction with cisplatin to enhance anticancer treatments.

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1.

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.

Replication of the lagging strand: a concert of at least 23 polypeptides.

DNA replication is one of the most important events in living cells, and it is still a key problem how the DNA replication machinery works in its details. A replication fork has to be a very dynamic apparatus since frequent DNA polymerase switches from the initiating DNA polymerase alpha to the processive elongating DNA polymerase delta occur at the leading strand (about 8 x 10(4) fold on both strands in one replication round) as well as at the lagging strand (about 2 x 10(7) fold on both strands in one replication round) in mammalian cells. Lagging strand replication involves a very complex set of interacting proteins that are able to frequently initiate, elongate and process Okazaki fragments of 180 bp. Moreover, key proteins of this important process appear to be controlled by S-phase check-point proteins. It became furthermore clear in the last few years that DNA replication cannot be considered uncoupled from DNA repair, another very important event for any living organism. The reconstitution of nucleotide excision repair and base excision repair in vitro with purified components clearly showed that the DNA synthesis machinery of both of these macromolecular events are similar and do share many components of the lagging strand DNA synthesis machinery. In this minireview we summarize our current knowledge of the components involved in the execution and regulation of DNA replication at the lagging strand of the replication fork.

Calf thymus Hsc70 and Hsc40 can substitute for DnaK and DnaJ function in protein renaturation but not in bacteriophage DNA replication.

Calf thymus (ct) Hsc70 has been shown previously to reactivate heat-inactivated prokaryotic and eukaryotic enzymes, while DnaK was able to reactivate solely prokaryotic enzymes. Here, we report on isolation from calf thymus of a DnaJ homolog, ctHsc40, and on testing of its cooperative function in three different assays: (i) reactivation of heat-inactivated DNA polymerases, (ii) stimulation of the ATPase activity of ctHsc70 chaperone, and (iii) replication of bacteriophage lambda DNA. Surprisingly, ctHsc70/ctHsc40 chaperones were found to reactivate the denatured prokaryotic and eukaryotic enzymes but not to promote bacteriophage lambda DNA replication, suggesting species specificity in DNA replication.

HIV-1 reverse transcriptase and integrase enzymes physically interact and inhibit each other.

Ordered molecular interactions and structural changes must take place within the human immunodeficiency virus type 1 (HIV-1) preintegration complex at various stages for successful viral replication. We demonstrate both physical and biochemical interactions between HIV-1 reverse transcriptase and integrase enzymes. This interaction may have implications on the in vivo functions of the two enzymes within the HIV-1 replication complex. It may be one of the various molecular interactions, which facilitate efficient HIV-1 replication within the target cells.

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.

The stereoselective targeting of a specific enzyme-substrate complex is the molecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors.

The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (+/-)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3'-azido-3'-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J. Med. Chem. 42, 4462-4470). The (+/-)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis. Only one enantiomer, (R)-(-)-PPO464, displayed antiviral activity against both the wild type and the K103N mutant HIV-1 RT and was found to interact exclusively with the reaction intermediate formed by RT complexed with both the DNA and the nucleotide substrates. Being the first compound of its class to display this behavior, (R)-(-)-PPO464 is the representative of a novel generation of nonnucleoside inhibitors. (R)-(-)-PPO464 showed significant synergism when tested in combination with other RT inhibitors and efficiently inhibited viral replication when tested against the laboratory strain HIV-1 IIIB or against either wild type or multidrug-resistant clinical isolates. Pharmacokinetic studies in mice and rats showed a more favorable profile for (R)-(-)-PPO464 than for the corresponding racemate. (R)-(-)-PPO464 was also found to easily cross the blood-brain barrier. The coadministration of the HIV-1 protease inhibitor ritonavir increased the bioavailability of (R)-(-)-PPO464, having little effect on its plasma and brain elimination rates.

Functional genomics in HIV-1 virus replication: protein-protein interactions as a basis for recruiting the host cell machinery for viral propagation.

Identification and characterization of protein-protein interactions between the host cell and parasites both enhance our understanding of basic cell biology and provide insights into central processes of parasite life cycles. Research on HIV-1 has broadened our knowledge of the various molecular events involved. However, our understanding of how this virus interacts with the host cell at the level of protein-protein interaction is still limited. Through these interactions the virus is able to recruit certain cellular metabolic pathways for its replication. Here we summarize our current knowledge of protein-protein interactions between HIV-1 and host cell factors during viral replication.

Anti-HIV-1 activity in vitro of ceftazidime degradation products.

Cephalosporins in aqueous solutions generate degradation products that inhibit in vitro HIV-1 replication in cell lines, as well as in primary cells (lymphocytes and macrophages). This effect is observed at concentrations that do not interfere with the normal functions of these cells. Upon chromatographic fractionation of an aqueous solution of hydrolysed ceftazidime, a high molecular weight fraction (MW 8000) with antiviral activity was isolated. The exact chemical nature of the active component responsible for the anti-HIV activity in vitro appears to be complex and is currently unknown. Inhibition of HIV-1 reverse transcriptase and RNase H activity was observed, however, higher concentrations than those needed to inhibit HIV replication were required. The inhibitory action of the hydrolysed ceftazidime was manifested during the early phase of the HIV-1 life-cycle. Despite a lack of a direct effect of the CD4/gp120 interaction, HIV-1 mediated cell fusion was inhibited by the hydrolysed ceftazidime, suggesting that the active principle acts in a very early stage of the viral life-cycle.

Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair.

The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system. Although XPA-RPA has been implicated in damage recognition, its activity in the DNA repair pathway remains controversial. By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak preference of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations. Further screening with artificially distorted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA. Conversely, RPA recognizes single-stranded sites but not backbone bending. Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA. The affinity of XPA for sharply bent duplexes, characteristic of architectural proteins, is not compatible with a direct function during recognition of nucleotide lesions. Instead, XPA in conjunction with RPA may constitute a regulatory factor that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly.

Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300.

We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1's DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.

A novel interaction of tRNA(Lys,3) with the feline immunodeficiency virus RNA genome governs initiation of minus strand DNA synthesis.

Complementarity between nucleotides at the 5' terminus of tRNA(Lys,3) and the U5-IR loop of the feline immunodeficiency virus RNA genome suggests a novel intermolecular interaction controls initiation of minus strand synthesis in a manner analogous to other retroviral systems. Base pairing of this tRNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome containing full-length or 5'-deleted variants of tRNA(Lys,3) hybridized to the primer-binding site. A major pause in RNA-dependent DNA synthesis occurred 14 nucleotides ahead of the primer-binding site with natural and synthetic tRNA(Lys,3) primers, indicating it was not a consequence of tRNA base modifications. The majority of the paused complexes resulted in dissociation of the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant whose 5' nucleotides are deleted relieved pausing at this position and subsequently allowed high level DNA synthesis. Additional experiments with tRNA-DNA chimeric primers were used to localize the stage of minus strand synthesis at which the tRNA-viral RNA interaction was disrupted. Finally, replacing nucleotides of the feline immunodeficiency virus U5-IR loop with the (A)(4) sequence of its human immunodeficiency virus (HIV)-1 counterpart also relieved pausing, but did not induce pausing immediately downstream of the primer-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequences immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.

Potentiation of inhibition of wild-type and mutant human immunodeficiency virus type 1 reverse transcriptases by combinations of nonnucleoside inhibitors and d- and L-(beta)-dideoxynucleoside triphosphate analogs.

Combinations of reverse transcriptase (RT) inhibitors are currently used in anti-human immunodeficiency virus therapy in order to prevent or delay the emergence of resistant virus and to improve the efficacy against viral enzymes carrying resistance mutations. Drug-drug interactions can result in either positive (additive or synergistic inhibition) or adverse (antagonistic interaction, synergistic toxicity) effects. Elucidation of the nature of drug interaction would help to rationalize the choice of antiretroviral agents to be used in combination. In this study, different combinations of nucleoside and nonnucleoside inhibitors, including D- and L-(beta)-deoxy- and -dideoxynucleoside triphosphate analogues, have been tested in in vitro RT assays against either recombinant wild-type RT or RT bearing clinically relevant nonnucleoside inhibitor resistance mutations (L100I, K103N, Y181I), and the nature of the interaction (either synergistic or antagonistic) of these associations was evaluated. The results showed that (i) synergy of a combination was not always equally influenced by the individual agents utilized, (ii) a synergistic combination could improve the sensitivity profile of a drug-resistant mutant enzyme to the single agents utilized, (iii) L-(beta)-enantiomers of nucleoside RT inhibitors were synergistic when combined with nonnucleoside RT inhibitors, and (iv) inter- and intracombination comparisons of the relative potencies of each drug could be used to highlight the different contributions of each drug to the observed synergy.

Replication protein A as a "fidelity clamp" for DNA polymerase alpha.

The current view of DNA replication in eukaryotes predicts that DNA polymerase alpha (pol alpha)-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol alpha elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol alpha displays a low processivity and, because of the lack of an intrinsic or associated 3'--> 5' exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol alpha-primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol alpha playing a dual role: (i) it stabilizes the pol alpha/primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol alpha. Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a "fidelity clamp" for pol alpha.

A coordinated interplay: proteins with multiple functions in DNA replication, DNA repair, cell cycle/checkpoint control, and transcription.

In eukaryotic cells, DNA transactions such as replication, repair, and transcription require a large set of proteins. In all of these events, complexes of more than 30 polypetides appear to function in highly organized and structurally well-defined machines. We have learned in the past few years that the three essential macromolecular events, replication, repair, and transcription, have common functional entities and are coordinated by complex regulatory mechanisms. This can be documented for replication and repair, for replication and checkpoint control, and for replication and cell cycle control, as well as for replication and transcription. In this review we cover the three different protein classes: DNA polymerases, DNA polymerase accessory proteins, and selected transcription factors. The "common enzyme-different pathway strategy" is fascinating from several points of view: first, it might guarantee that these events are coordinated; second, it can be viewed from an evolutionary angle; and third, this strategy might provide cells with backup mechanisms for essential physiological tasks.

In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding.

Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease with important functions in DNA replication and DNA repair. It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro. The PCNA interaction site is located close to the C terminus of Fen1 and is flanked by a conserved basic region of 35-38 amino acids in eukaryotic species but not in archaea. We have constructed two deletion mutants of human Fen1 that lack either the PCNA interaction motif or a part of its adjacent C-terminal region and analyzed them in a variety of assays. Remarkably, deletion of the basic C-terminal region did not affect PCNA interaction but resulted in a protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe defect in substrate binding. Our results suggest that the C terminus of eukaryotic Fen1 consists of two functionally distinct regions that together might form an important regulatory domain.