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Biomedical subjects

Giovanni Maga

Publications and source records attributed to Giovanni Maga.

At least 19 recordsLinked to original sources
PubMed2003

Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners.

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.

Cells, Cultured↗
PubMed2003

Proliferating cell nuclear antigen (PCNA): a dancer with many partners.

Proliferating cell nuclear antigen (PCNA) was originally characterised as a DNA sliding clamp for replicative DNA polymerases and as an essential component of the eukaryotic chromosomal DNA replisome. Subsequent studies, however, have revealed its striking ability to interact with multiple partners, which are involved in several metabolic pathways, including Okazaki fragment processing, DNA repair, translesion DNA synthesis, DNA methylation, chromatin remodeling and cell cycle regulation. PCNA in mammalian cells thus appears to play a key role in controlling several reactions through the coordination and organisation of different partners. Two major questions have emerged: how do these proteins access PCNA in a coordinated manner, and how does PCNA temporally and spatially organise their functions? Structural and biochemical studies are starting to provide a first glimpse of how both tasks can be achieved.

Animals↗
PubMed2003

Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

Several proteins involved in DNA synthesis are part of the so-called 'replication factories' that are anchored on non-chromatin nuclear structures. We report here that human kin17, a nuclear stress-activated protein, associates with both chromatin and non-chromatin nuclear structures in a cell cycle- and DNA damage-dependent manner. After L-mimosine block and withdrawal we observed that kin17 protein was recruited in the nucleus during re-entry and progression through S phase. These results are consistent with a role of kin17 protein in DNA replication. About 50% of the total amount of kin17 protein was detected on nuclear structures and could not be released by detergents. Furthermore, the amount of kin17 protein greatly increased in both G(1)/S and S phase-arrested cells in fractions containing proteins anchored to nuclear structures. The detection of kin17 protein showed for the first time its preferential assembly within non-chromatin nuclear structures in G(1)/S and S phase-arrested cells, while the association with these structures was found to be less stable in the G(2)/M phase, as judged by fractionation of human cells and immunostaining. In asynchronous growing cells, kin17 protein interacted with both chromatin DNA and non-chromatin nuclear structures, while in S phase-arrested cells it interacted mostly with non-chromatin nuclear structures, as judged by DNase I treatment and in vivo UV cross-linking. In the presence of DNA damage in S phase cells, the distribution of kin17 protein became mainly associated with chromosomal DNA, as judged by limited formaldehyde cross-linking of living cells. The physical interaction of kin17 protein with components of the nuclear matrix was confirmed and visualized by indirect immunofluorescence and immunoelectron microscopy. Our results indicate that, during S phase, a fraction of the human kin17 protein preferentially associates with the nuclear matrix, a fundamentally non-chromatin higher order nuclear structure, and to chromatin DNA in the presence of DNA damage.

Cell Cycle↗
PubMed2003

Nevirapine-selected mutations Y181I/C of HIV-1 reverse transcriptase confer cross-resistance to stavudine.

Stavudine administration did not increase the frequency of Y181I/C reverse transcriptase (RT) mutations in non-nucleoside reverse transcriptase inhibitor (NNRTI)-treated patients. However, recombinant Y181C HIV-1 showed reduced stavudine susceptibility with respect to both recombinant wild-type and K103N HIV-1 strains. In addition, recombinant Y181I RT enzyme showed reduced susceptibility to stavudine with respect to both wild-type and K103N RT. A previously unnoticed role of Y181I/C RT changes selected by nevirapine or other NNRTI in determining stavudine resistance is documented.

Antiretroviral Therapy, Highly Active↗
PubMed2003

Human DNA polymerase lambda diverged in evolution from DNA polymerase beta toward specific Mn(++) dependence: a kinetic and thermodynamic study.

The recently discovered human DNA polymerase lambda (DNA pol lambda) has been implicated in translesion DNA synthesis across abasic sites. One remarkable feature of this enzyme is its preference for Mn(2+) over Mg(2+) as the activating metal ion, but the molecular basis for this preference is not known. Here, we present a kinetic and thermodynamic analysis of the DNA polymerase reaction catalyzed by full length human DNA pol lambda, showing that Mn(2+) favors specifically the catalytic step of nucleotide incorporation. Besides acting as a poor coactivator for catalysis, Mg(2+) appeared to bind also to an allosteric site, resulting in the inhibition of the synthetic activity of DNA pol lambda and in an increased sensitivity to end product (pyrophosphate) inhibition. Comparison with the closely related enzyme human DNA pol beta, as well as with other DNA synthesising enzymes (mammalian DNA pol alpha and DNA pol delta, Escherichia coli DNA pol I, and HIV-1 reverse transcriptase) indicated that these features are unique to DNA pol lambda. A deletion mutant of DNA pol lambda, which contained the highly conserved catalytic core only representing the C-terminal half of the protein, showed biochemical properties comparable to the full length enzyme but clearly different from the close homologue DNA pol beta, highlighting the existence of important differences between DNA pol lambda and DNA pol beta, despite a high degree of sequence similarity.

Allosteric Site↗
PubMed2003

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions.

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.

Base Sequence↗
PubMed2003

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

Codon↗
PubMed2003

DNA replication: a complex matter.

In eukaryotic cells, the essential function of DNA replication is carried out by a network of enzymes and proteins, which work together to rapidly and accurately duplicate the genetic information of the cell. Many of the components of this DNA replication apparatus associate with other cellular factors as components of multiprotein complexes, which act cooperatively in networks to regulate cell cycle progression and checkpoint control, but are distinct from the pre-replication complexes that associate with the origins and regulate their firing. In this review, we summarize current knowledge about the composition and dynamics of these large multiprotein complexes in mammalian cells and their relationships to the replication factories.

Animals↗
PubMed2003

Antivirals at the mirror: the lack of stereospecificity of some viral and human enzymes offers novel opportunities in antiviral drug development.

The enantioselectivity of enzymes, namely the property of enzymes to recognise and metabolise only one of the two enantiomers of chiral molecules, is related to the chiral structure of the enzymes, reflecting the three-dimensional folding of the polypeptide backbone and the orientation of the amino acid side chains in the folded molecule. Because of the chirality of the amino acids (L), the chemistry of life should be highly sensitive to different enantiomers of chiral substrates. However, in a world consisting only of D-nucleosides and L-amino acids, an enzyme which lacks enantio-selectivity does not reduce its fitness, since there is no chance of molecular misunderstanding when no other choice is available. Thus, although enantioselectivity is theoretically essential for life we do not expect to be always present among the biochemical properties of enzymes. If this is the case for key enzymes involved in virus infection or cancer, how to exploit such lack of enantioselectivity for a novel approach to antiviral or anticancer chemotherapy? The present review will discuss the possible lack of enantioselectivity of enzymes and its relevance for the developing of novel drugs with the inverted optical configuration.

Antiviral Agents↗
PubMed2002

Human DNA polymerase lambda functionally and physically interacts with proliferating cell nuclear antigen in normal and translesion DNA synthesis.

Proliferating cell nuclear antigen (PCNA) has been shown to interact with a variety of DNA polymerases (pol) such as pol delta, pol epsilon, pol iota, pol kappa, pol eta, and pol beta. Here we show that PCNA directly interacts with the newly discovered pol lambda cloned from human cells. This interaction stabilizes the binding of pol lambda to the primer template, thus increasing its affinity for the hydroxyl primer and its processivity in DNA synthesis. However, no effect of PCNA was detected on the rate of nucleotide incorporation or discrimination efficiency by pol lambda. PCNA was found to stimulate efficient synthesis by pol lambda across an abasic (AP) site. When compared with pol delta, human pol lambda showed the ability to incorporate a nucleotide in front of the lesion. Addition of PCNA led to efficient elongation past the AP site by pol lambda but not by pol delta. However, when tested on a template containing a bulky DNA lesion, such as the major cisplatin Pt-d(GpG) adduct, PCNA could not allow translesion synthesis by pol lambda. Our results suggest that the complex between PCNA and pol lambda may play an important role in the bypass of abasic sites in human cells.

Base Sequence↗
PubMed2002

Hepatitis C virus NS3 ATPase/helicase: an ATP switch regulates the cooperativity among the different substrate binding sites.

The protease/helicase NS3 is believed to play a central role in the replication cycle of the hepatitis C virus (HCV), and, therefore, it is an attractive target for antiviral chemotherapy. Several enzymological studies and crystallographic structures are available for the NS3 protease and helicase domains individually, but less is known about the NTPase and helicase activities of the full-length protein. The aim of our study was to characterize from an enzymological point of view the mechanism of interaction of the full-length NS3 protease/helicase with its nucleic acid (NA) and ATP substrates. Our kinetic analysis revealed that both the NA and ATP substrates can interact cooperatively with the enzyme through the coordinated action of two binding sites. Moreover, the observation of a reciprocal influence of both substrates on the kinetics of their interaction with the enzyme suggested that the NS3 helicase works as a dimer which can exist in three functionally different states: (i) an unbound state, with two equivalent low-affinity binding sites for ATP, which shows cooperative high-affinity NA binding; (ii) an ATP-bound state, with two equivalent low-affinity NA binding sites; and (iii) a NA-bound state, with two equivalent high-affinity ATP binding sites. The cycling between these different conformational states is thus regulated by an ATP switch. These results are discussed in light of the current models for NA unwinding by the HCV NS3 helicase.

Adenosine Triphosphatases↗
PubMed2002

DNA polymerase theta purified from human cells is a high-fidelity enzyme.

With the aim to identify unconventional DNA polymerases from human cells, we have set up a special assay to fractionate HeLa extracts based on the ability (i) to bypass DNA lesions, (ii) to be resistant to aphidicolin and an inhibitory antibody against pol alpha and (iii) to be non-responsive to proliferating cell nuclear antigen. After eight different chromatographic steps, an aphidicolin-resistant DNA polymerase activity was obtained that was able to utilize either undamaged or abasic sites-containing DNA with the same efficiency. Biochemical characterization and immunoblot analysis allowed its identification as the human homologue of DNA polymerase theta (hpol theta), whose cDNA has been cloned by homology with the mus308 gene of Drosophila melanogaster but still awaited detailed biochemical characterization. The purified hpol theta was devoid of detectable helicase activity, possessed a 3'-->5' exonuclease activity and showed biochemical properties clearly distinct from any other eukaryotic DNA polymerase known so far. Misincorporation and fidelity assays showed that: (i) hpol theta was able to catalyze efficiently DNA synthesis past an abasic site; and (ii) hpol theta showed high fidelity. Our findings are discussed in light of the proposed physiological role of hpol theta.

DNA↗
PubMed2002

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins.

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.

Animals↗
PubMed2002

DNA polymerase lambda from calf thymus preferentially replicates damaged DNA.

A new gene (POLL), has been identified encoding the novel DNA polymerase lambda and mapped to mouse chromosome 19 and at human chromosome 10. DNA polymerase lambda contains all the critical residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis of DNA polymerization and has been assigned to family X based on sequence homology with polymerase beta, lambda, mu, and terminal deoxynucleotidyltransferase. Here we describe a purification of DNA polymerase lambda from calf thymus that preferentially can replicate damaged DNA. By testing polymerase activity on non-damaged and damaged DNA, DNA polymerase lambda was purified trough five chromatographic steps to near homogeneity and identified as a 67-kDa polypeptide that cross-reacted with monoclonal antibodies against DNA polymerase beta and polyclonal antibodies against DNA polymerase lambda. DNA polymerase lambda had no detectable nuclease activities and, in contrast to DNA polymerase beta, was aphidicolin-sensitive. DNA polymerase lambda was a 6-fold more accurate enzyme in an M13mp2 forward mutation assay and 5-fold more accurate in an M13mp2T90 reversion system than human recombinant DNA polymerase beta. The biochemical properties of the calf thymus DNA polymerase lambda, described here for the first time, are discussed in relationship to the proposed role for this DNA polymerase in vivo.

Animals↗
PubMed2002

A new and efficient synthesis of substituted 6-[(2'-dialkylamino)ethyl] pyrimidine and 4-N,N-dialkyl-6-vinyl-cytosine derivatives and evaluation of their anti-rubella activity.

New 6-[(2'-dialkylamino)ethyl]-4(3H)-pyrimidinones were prepared by a multistep procedure starting from acetone dicarboxylic acid diethyl ester and urea derivatives. These compounds were used as starting materials to obtain 4-N,N-dialkyl-6-vinyl-pyrimidine derivatives by an unprecedented tandem C-6 side chain Hofmann-like elimination/C-4 pyrimidinone substitution. Among the new derivatives obtained, various compounds show anti-Rubella activity. The inhibition of HIV-1 Reverse Transcriptases (RT), from both wild type and modified viruses, is also reported.

Antiviral Agents↗
PubMed2002

Non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors: past, present, and future perspectives.

Along with nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive and important place in the treatment of HIV-1 infections, and are in rapid development. These compounds can be grouped into two classes: the first generation NNRTIs, mainly discovered by random screening, and the second generation NNRTIs, developed as a result of comprehensive strategies involving molecular modelling, rationale-based drug synthesis, biological and pharmacokinetic evaluations. The recent boom of NNRTIs is mainly due to their antiviral potency, high specificity and low toxicity. The rapid emergence of drug-resistant HIV-1 strains induced by the first generation drugs is a disadvantage bypassed, in part, by the broad spectrum second generation NNRTIs. Starting from the first generation, this review will focus on the second generation NNRTIs dealing with the recent and most interesting published results, highlighting the guidelines for the development of a third generation of NNRTIs.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Combinations against combinations: associations of anti-HIV 1 reverse transcriptase drugs challenged by constellations of drug resistance mutations.

The reverse transcriptase inhibitors still represent the majority of the clinically used anti-HIV drugs and constitute the main backbone of currently employed combinatorial regimens. A major obstacle to successfull chemotherapic eradication of HIV is the emergence of viral strains resistant to the drugs in use. Counteracting the emergence of resistance necessitates alternating the panel of agents employed. In order to rationally design alternative drug combinations, physicians not only must know the genotype of the emerging viral strains, but should also be able to correlate it with its resistant phenotype. However, resistant viral strains usually carry multiple mutations, whose reciprocal influences on the overall level of resistance are largely unknown. Moreover, the choice of agents to be combined must take in account drug-drug interactions and adverse metabolic effects. This review will outline the main pharmacological and clinical features of the currently utilised anti-reverse transcriptase drugs, as well as the correspondent resistance profiles selected during therapy. A major focus will be on the reciprocal influence of drug associations on their own metabolism as well as on the interacting effects of the selected combinations of drug resistance mutations.

Animals↗