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Biomedical subjects

Ulrich Hübscher

Publications and source records attributed to Ulrich Hübscher.

At least 19 recordsLinked to original sources
PubMed2003

DNA polymerase beta can incorporate ribonucleotides during DNA synthesis of undamaged and CPD-damaged DNA.

Overexpression of the error-prone DNA polymerase beta (Pol beta) has been found to increase spontaneous mutagenesis by competing with the replicative polymerases during DNA replication. Here, we investigate an additional mechanism potentially used by Pol beta to enhance genetic instability via its ability to incorporate ribonucleotides into DNA. By using an in vitro primer extension assay, we show that purified human and calf thymus Pol beta can synthesize up to 8-mer long RNA. Moreover, Pol beta can efficiently incorporate rCTP opposite G in the absence of dCTP and, to a lesser extent, rATP opposite T in the absence of dATP and rGTP opposite C in the absence of dGTP. Recently, Pol beta was shown to catalyze in vitro translesion replication of a thymine cyclobutane pyrimidine dimer (CPD). Here, we investigate if ribonucleotides could be incorporated opposite the CPD damage and modulate the efficiency of the bypass process. We find that all four rNTPs can be incorporated opposite the CPD lesion, and that this process affects translesion synthesis. We discuss how incorporation of ribonucleotides into DNA may contribute to the high frequency of mutagenesis observed in Pol beta up-regulating cells.

Animals↗
PubMed2003

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation.

Cyclin-dependent kinase (Cdk) Cdk1-Cyclin A can phosphorylate Flap endonuclease 1 (Fen1), a key-enzyme of the DNA replication machinery, in late S phase. Cdk1-cyclin A forms a complex in vitro and in vivo with Fen1. Furthermore, Fen1 phosphorylation is detected in vivo and depends upon Cdks activity. As a functional consequence of phosphorylation by Cdk1-Cyclin A in vitro, endo- and exonuclease activities of Fen1 are reduced whereas its DNA binding is not affected. Moreover, phosphorylation of Fen1 by Cdk1-Cyclin A abrogates its proliferating cell nuclear antigen (PCNA) binding thus preventing stimulation of Fen1 by PCNA. Concomitantly, human cells expressing the S187A mutant defective for Cdk1-Cyclin A phosphorylation accumulate in S phase consistent with a failure in cell cycle regulation through DNA replication. Our results suggest a novel regulatory role of Cdks onto the end of S phase by targeting directly a key enzyme involved in DNA replication.

Adenosine Triphosphate↗
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

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA.

The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified deoxyribonucleoside 5'-triphosphates (dNTPs) covering all four naturally occurring nucleobases for potential use in DNA modification. A total of 30 modified dNTPs with either fluorescent or non-fluorescent reporter group attachments were systematically evaluated individually and in combinations for high-density incorporation using different model and natural DNA templates. Furthermore, we show a side-by-side comparison of the incorporation efficiencies of a family A (Taq) and B (Vent(R) exo-) type DNA polymerase using the differently modified dNTP substrates. Our results show superior performance by a family B-type DNA polymerase, Vent(R) exo-, which is able to fully synthesize a 300 bp DNA product when all natural dNTPs are completely replaced by their biotin-labeled dNTP analogs. Moreover, we present systematic testing of various combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling of DNA with up to four differently modified dNTPs.

Base Sequence↗
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

The acetylatable lysines of human Fen1 are important for endo- and exonuclease activities.

Human Fen1 can be acetylated in vivo and in vitro resulting in reduced endonuclease and exonuclease activities in vitro. Acetylation occurs at four lysines located at the C terminus of Fen1, which is important for DNA binding. In this paper we show that Fen1 mutant proteins lacking the lysines at the C terminus have both reduced PCNA independent exonucleolytic and endonucleolytic activities. However, lysines at the C terminus are not required for PCNA stimulation of human Fen1. A double flap substrate was optimal for human Fen1 endonuclease and did not require the C-terminal lysines. Similarly, a one nucleotide 3'-overhang nick substrate was optimal for human Fen1 exonuclease and also did not require the C-terminal lysines. Finally, we found by an electromobility shift assay that human Fen1 had a different mode of binding with a double flap substrate containing a one nucleotide 3'-tail when compared to various other flap substrates. Taken together, our results confirm the double flap substrate as the likely in vivo intermediate for human Fen1 and that the C-terminal lysines are important for the endonuclease and exonuclease activities likely through DNA binding.

Acetylation↗
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

Multiple roles for kinases in DNA replication.

DNA replication is carried out by the replisome, which includes several proteins that are targets of cell-cycle-regulated kinases. The phosphorylation of proteins such as replication protein A, DNA polymerase-alpha and -delta, replication factor C, flap endonuclease 1 and DNA ligase I leads to their inactivation, suggesting that phosphorylation is important in the prevention of re-replication. Moreover, the phosphorylation of several of these replication proteins has been shown to block their association with the 'moving platform'-proliferating cell nuclear antigen. Therefore, phosphorylation seems to be a crucial regulator of replisome assembly and DNA replication, although its precise role in these processes remains to be clarified.

Animals↗
PubMed2002

Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

Adenosine Triphosphatases↗
PubMed2002

The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair.

The conserved, structure-specific flap endonuclease FEN1 cleaves 5' DNA flaps that arise during replication or repair. To address in vivo mechanisms of flap cleavage, we developed a screen for human FEN1 mutants that are toxic when expressed in yeast. Two targets were revealed: the flexible loop domain and the catalytic site. Toxic mutants caused G(2) arrest and cell death and were unable to repair methyl methanesulfonate lesions. All the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5' flaps, the loop mutants exhibited partial ability to cut 5' flaps when an adjacent single nucleotide 3' flap was present. We suggest that the flexible loop is important for efficient cleavage through positioning the 5' flap and the catalytic site.

Base Sequence↗
PubMed2002

Colocalization of human Rad17 and PCNA in late S phase of the cell cycle upon replication block.

In response to replication block or DNA damage in S phase the DNA replication and DNA damage checkpoints are activated. The current model in human predicts, that a Rad17/Replication factor C (RF-C) complex might serve as a recruitment complex for the Rad9/Hus1/Rad1 complex to sites of replication block or DNA damage. In this study we have investigated the fate of the Rad17/RF-C complex after treatment of synchronized Hela cells with the replication inhibitor hydroxyurea. In hydroxyurea treated cells the RF-C p37 subunit became more resistant to extraction. Moreover, co-immunoprecipitation studies with extracts of hydroxyurea treated cells showed an interaction of RF-C p37 with Rad17 and of PCNA with Rad9 and RF-C p37. An enhanced colocalization of Rad17 and PCNA in late S phase after hydroxyurea treatment was observed. Our data suggested, that upon replication block a Rad17/RF-C complex is recruited to sites of DNA lesions in late S phase, binds the Rad9/Hus1/Rad1 complex and enables it to interact with PCNA. An interaction of Rad17/RF-C with PCNA appears to be mediated by the small RF-C p37 subunit, suggesting that PCNA might provide communication between replication checkpoint control and DNA replication and repair.

4-Nitroquinoline-1-oxide↗
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

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

The TREX2 3'-->5' exonuclease physically interacts with DNA polymerase delta and increases its accuracy.

Proofreading function by the 3'-->5' exonuclease of DNA polymerase delta (pol delta) is consistent with the observation that delta efficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol delta from isotonic (pol delta(i)) and detergent (pol delta(d)) calf thymus extracts. Pol delta had a 20-fold higher ratio of exonuclease to DNA polymerase than pol deltai. This was due to the physical association of the TREX2 3'-->5' exonuclease to pol delta(d), which was missing from pol delta(i). Pol delta(d) was fivefold more accurate than pol i under error-prone conditions (1 mM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate than pol delta(i) under error-prone conditions (1 mM dGTP and 20 microM dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 microM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3' --> 5' exonucleases, such as TREX2, through its association with pol delta can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

Animals↗