Giovanni Maga | Consiglio Nazionale delle Ricerche (CNR) (original) (raw)

Papers by Giovanni Maga

Biochemistry, 1995

Kinetic analysis of DNA polymerase epsilon in its interaction with the homopolymeric template-pri... more Kinetic analysis of DNA polymerase epsilon in its interaction with the homopolymeric template-primer poly(dA)/oligo(dT) and a singly-primed synthetic oligonucleotide of defined sequence indicated that primer utilization is inhibited by single-stranded DNA. Long single-stranded DNA regions appear to sequester DNA polymerase epsilon via nonproductive binding, thus reducing its catalytic efficiency. Proliferating cell nuclear antigen can reduce this nonproductive effect by increasing the rate of primer binding by DNA polymerase epsilon. Once the complex between DNA polymerase epsilon and the primer is formed, proliferating cell nuclear antigen can increase the rate of nucleotide incorporation. The results suggested a dual role of proliferating cell nuclear antigen in stimulating the activity of DNA polymerase epsilon, namely, first to facilitate primer binding and second to stimulate the synthetic activity itself. A model for the interaction between these two proteins in DNA synthesis is discussed.

Journal of Cell Science, 2003

Proliferating cell nuclear antigen (PCNA) was originally characterised as a DNA sliding clamp for... more 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.

Journal of Medicinal Chemistry, 2006

Molecular modeling studies and an updated highly predictive 3-D QSAR model led to the discovery o... more Molecular modeling studies and an updated highly predictive 3-D QSAR model led to the discovery of exceptionally potent indolyl aryl sulfones (IASs) characterized by the presence of either a pyrrolidyn-2-one nucleus at the indole-2-carboxamide or some substituents at the indole-2-carbohydrazide. Compounds 7 and 9 were found active in the sub-nanomolar range of concentration in both MT-4 and C8166 cell-based anti-HIV assays. These compounds, and in particular compound 9, also showed excellent inhibitory activity against both HIV-112 and HIV-AB1 primary isolates in lymphocytes and against HIV WT in macrophages.

Journal of Medicinal Chemistry, 2007

Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-ox... more Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-oxopyrimidines (DABOs, S-DABOs, and NH-DABOs) as potent and selective anti-HIV-1 agents belonging to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class, we report here the synthesis and biological evaluation of a novel series of DABOs bearing a N,N-disubstituted amino group or a cyclic amine at the pyrimidine-C2 position, a hydrogen atom or a small alkyl group at C5 and/or at the benzylic position, and the favorable 2,6-difluorobenzyl moiety at the C6 position (F2-N,N-DABOs). The new compounds were highly active up to the subnanomolar level against both wt HIV-1 and the Y181C mutant and at the submicromolar to nanomolar range against the K103N and Y188L mutant strains. Such derivatives were more potent than S-DABOs, NH-DABOs, and nevirapine and efavirenz were chosen as reference drugs. The higher inhibitor adaptability to the HIV-1 RT non-nucleoside binding site (NNBS) may account for the higher inhibitory effect exerted by the new molecules against the mutated RTs.

Bioorganic & Medicinal Chemistry, 2005

2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F 2 -NH-DAB... more 2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F 2 -NH-DABOs) 4, 5 belonging to the dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family and bearing different alkyl-and arylamino side chains at the C 2 -position of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the nonnucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1 strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C 2 -amino side chain, but it associated with the presence of one or two methyl groups (one at the pyrimidine C 5 -position and the other at the benzylic carbon), being thymine, a-methyluracil or a-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cytopathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-a-methylthymine derivatives 4d, 5h 0 -n 0 showed the highest potency and selectivity among tested compounds, both a properly sized C 2 -NH side chain and the presence of two methyl groups (at C 5 and benzylic positions) being crucial for high antiviral action.

Virology, 2004

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activ... more The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5V -triphosphatase (NTPase), and RNA 5V -triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DNS3 is Mg 2+ -dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg 2+ . The R513A substitution in the C-terminal domain of DNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg 2+ -dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain. D

Journal of Molecular Biology, 2003

DNA polymerase l is a novel enzyme of the family X of DNA polymerases. The recent demonstration o... more DNA polymerase l is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5 0 -deoxyribose-5 0 -phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase b suggested a novel b-like enzyme. All these properties support a role of DNA polymerase l in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase l are still largely unknown. Here we give evidence that human DNA polymerase l has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3 0 OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase l efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase l might suggest additional roles for this enzyme in DNA replication and repair processes.

Biochemistry, 1996

By using a complementation assay for a replication factor C dependent DNA polymerase activity on ... more By using a complementation assay for a replication factor C dependent DNA polymerase activity on a singly-primed M13 DNA template, we have isolated from calf thymus a multiprotein complex active in DNA replication. For this, the inclusion of ATP during the entire isolation procedure was essential, since the complex decayed after omission of ATP. This complex contains at least DNA polymerase alpha/primase, DNA polymerase delta, and replication factor C as shown by gel-filtration and coimmunoprecipitation experiments. It is functionally active in replication of primed and unprimed single-stranded M13 DNA templates. Furthermore, in the presence of proliferating cell nuclear antigen and ATP, it forms an isolatable holoenzyme/template-primer complex. Replication factor C apparently mediates the interaction of DNA polymerase delta in the complex with proliferating cell nuclear antigen, through an ATP-dependent mechanism. This interaction appears to stabilize the binding of the complex to a template-primer and to coordinate the activity of DNA polymerase alpha/primase and DNA polymerase delta during replication of a single-stranded DNA template. Our data suggest the existence of an asymmetric DNA polymerase complex in mammalian cells.

Nature, 2007

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells 1 . Auxiliar... more Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells 1 . Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols-belonging to the B, Y and X classes-during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential 2-4 . A major and specific effect was found for 8-oxo-G bypass with DNA pols l and g. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol l, and 68fold by DNA pol g, respectively. Experiments with DNA-pol-lnull cell extracts suggested an important role for DNA pol l. On the other hand, DNA pol i, together with DNA pols a, d and b, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.

Febs Letters, 2001

Ordered molecular interactions and structural changes must take place within the human immunodefi... more 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. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Biochemistry, 2002

The protease/helicase NS3 is believed to play a central role in the replication cycle of the hepa... more 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.

Antimicrobial Agents and Chemotherapy, 2004

The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the hu... more The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.

Journal of Medicinal Chemistry, 1995

Two series of selective inhibitors of herpes simplex virus types 1 and 2 (HSV1,2) thymidine kinas... more Two series of selective inhibitors of herpes simplex virus types 1 and 2 (HSV1,2) thymidine kinases (TK) have been developed as potential treatment of recurrent virus infections. Among compounds related to the potent base analog W-[m-(trifluoromethyl)phenyl]guanine (mCF3-@ Abstract published in Advance ACS Abstracts, December 1, 1994. rine eye7 and ear8 models for HSV1, rabbitg and squirrel monkeylo eye models for HSV1, and a guinea pig vaginal model for HSV2.11 In these models both spontaneous and induced viral reactivation can be studied. Two studies of TK inhibitors on spontaneous HSV recurrences in vivo have been reported. 5'-Ethynylthymidine was given intraperitoneally (ip) as an aqueous suspension to squirrel monkeys in which latent infections had been established by prior corneal inoculation with HSV1.IO After 25 days of dosing, viral lesions in the corneas of animals treated with drug were compared with those of control animals. Recurrent lesions were found in 3/20 eyes of treated animals compared to 8/20 eyes of control animals (P = 0.077).1° In another study,

Expert Opinion on Investigational Drugs, 1998

As a general rule, enzymes act on only one enantiomer of a chiral substrate and only one of the e... more As a general rule, enzymes act on only one enantiomer of a chiral substrate and only one of the enantiomeric forms of a chiral molecule may bind effectively at the catalytic site, displaying biological activity. In recent years, some exceptions have been found among viral and cellular enzymes involved in the synthesis of deoxynucleoside triphosphates and in their polymerisation into DNA. Examples are: herpes virus thymidine kinases, cellular deoxycytidine kinase and deoxynucleotide kinases, human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, hepatitis B virus (HBV) DNA polymerase and, to a lesser extent, some cellular DNA polymerases. The lack of enantioselectivity allows herpes simplex virus (HSV) thymidine kinase and cellular deoxycytidine kinase to phosphorylate the unnatural L-beta-enantiomers of D-thymidine and D-deoxycytidine, respectively, or of their analogues to monophosphate. This phosphorylation represents the first and often the rate-limiting step of their activation to triphosphates. The L-triphosphates can then exert antiviral (anti-HSV, anti-Human cytomegalovirus, anti-HIV-1, anti-HBV) and anticancer activities. Although only one L-nucleoside (3TC) has so far gained United States of America Food and Drug Administration (USA FDA) approval for clinical use against HIV-1, other L-enantiomers of nucleoside analogues, which have shown antiviral or anticancer activity in cell cultures are in clinical trials. Their resistance to enantioselective enzymes, such as thymidine phosphorylase, thymidylate synthase, (deoxy)-cytidine and dCMP deaminases, and their lower affinity for the mitochondrial thymidine kinase can ensure a higher selectivity and lower cytotoxicity with respect to those exerted by their corresponding natural D-enantiomers and might be exploited to solve problems arising during chemotherapy, such as metabolic inactivation, cytotoxicity and drug-resistance.

Journal of Molecular Biology, 1997

The kinetic parameters governing the inhibition by Nevirapine of the RNA-dependent DNA synthesis ... more The kinetic parameters governing the inhibition by Nevirapine of the RNA-dependent DNA synthesis catalyzed by HIV-1 reverse transcriptase have been determined by steady-state kinetic analysis with the wild-type enzyme and with mutant reverse transcriptases containing the single amino acid substitutions L100I, K103N, V106A, V179D, Y181I and Y188L. While the mutant V179D was inhibited by Nevirapine as the wild-type enzyme, all the other mutations displayed a 17 to 90-fold reduced sensitivity to the drug in the order: Y181I < (i.e. less sensitive) Y188L < V106A < L100I < K103N < wild-type. Determination of the rate constants for Nevirapine binding (k on ) and dissociation (k off ) for the mutant and wildtype enzymes showed that mutations L100I and V106A increased the k off values by 12 and 8.5-fold, respectively, without signi®cantly affecting the k on , whereas mutation K103N decreased the k on 5-fold without increasing the k off . Mutations Y181I and Y188L, on the other hand, conferred resistance to Nevirapine affecting both k off and k on values. In addition, mutations L100I and Y181I reduced the catalytic potential of HIV-1 RT. Thus, Nevirapine resistance could arise from a combination of loss of stabilizing interactions and emergence of steric and thermodynamic barriers for drug binding, depending on the particular amino acid substitution involved.

Proceedings of The National Academy of Sciences, 2001

DNA polymerase (pol) ␦ is essential for both leading and lagging strand DNA synthesis during chro... more DNA polymerase (pol) ␦ is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol ␦ has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA͞DNA 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 RNA͞DNA primer, we could measure strand displacement by pol ␦ coupled to DNA synthesis. Our results suggested that pol ␦ 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 ␦, 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 ␦ is modulated by the concerted action of PCNA, RP-A and Fen 1.

Annual Review of Biochemistry, 2002

f Abstract Any living cell is faced with the fundamental task of keeping the genome intact in ord... more f Abstract Any living cell is faced with the fundamental task of keeping the genome intact in order to develop in an organized manner, to function in a complex environment, to divide at the right time, and to die when it is appropriate. To achieve this goal, an efficient machinery is required to maintain the genetic information encoded in DNA during cell division, DNA repair, DNA recombination, and the bypassing of damage in DNA. DNA polymerases (pols) ␣, ␤, ␥, ␦, and ⑀ are the key enzymes required to maintain the integrity of the genome under all these circumstances. In the last few years the number of known pols, including terminal transferase and telomerase, has increased to at least 19. A particular pol might have more than one functional task in a cell and a particular DNA synthetic event may require more than one pol, which suggests that nature has provided various safety mechanisms. This multi-functional feature is especially valid for the variety of novel pols identified in the last three years. These are the lesion-replicating enzymes pol , pol , pol , pol , and Rev1, and a group of pols called pol , pol , pol , pol , and pol that fulfill a variety of other tasks.

Biochemistry, 2003

The recently discovered human DNA polymerase lambda (DNA pol lambda) has been implicated in trans... more 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.

Journal of Molecular Biology, 2002

With the aim to identify unconventional DNA polymerases from human cells, we have set up a specia... more 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 a 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 u (hpol u), whose cDNA has been cloned by homology with the mus308 gene of Drosophila melanogaster but still awaited detailed biochemical characterization. The purified hpol u was devoid of detectable helicase activity, possessed a 3 0 ! 5 0 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 u was able to catalyze efficiently DNA synthesis past an abasic site; and (ii) hpol u showed high fidelity. Our findings are discussed in light of the proposed physiological role of hpol u q

Antimicrobial Agents and Chemotherapy, 2000