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U Hübscher

Publications and source records attributed to U Hübscher.

At least 55 records · Page 3Linked to original sources
PubMed1998

[DNA replication: mechanism and significance for general practice].

The maintenance of genetic information in a cell is ensured by essential macromolecular events called DNA replication and DNA repair. The cell possesses many control mechanisms that allow the optimal strategy under extreme physiological conditions. A cell that is in the process of replicating its DNA and faces a genotoxic stress such as UV-damage, X-ray damage and influences of mutagens will turn off DNA replication and instead turn on DNA repair mechanisms. We have learned in the last three years that in all these processes identical DNA synthetic enzymes are involved. They are, however, coordinated to the actually required metabolic events through a ring-shaped protein called proliferating cell nuclear antigen (PCNA). Exact knowledge of these protein-protein interactions will eventually open up novel therapeutic strategies against cancer.

Cell Transformation, Neoplastic↗
PubMed1998

Mammalian base excision repair by DNA polymerases delta and epsilon.

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.

Animals↗
PubMed1998

DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by these two essential DNA polymerases. DNA ligase I inhibited replication factor C-independent DNA synthesis by polymerase delta. Our results suggest that the inhibition may be due to DNA ligase I interaction with proliferating cell nuclear antigen (PCNA) and not to a direct interaction with the DNA polymerase delta itself. Strand displacement activity by DNA polymerase delta was also affected by DNA ligase I. The DNA polymerase delta holoenzyme (composed of DNA polymerase delta, PCNA, and replication factor C) was inhibited in the same way as the DNA polymerase delta core, strengthening the hypothesis of a PCNA interaction. Contrary to DNA polymerase delta, DNA synthesis by DNA polymerase epsilon was stimulated by DNA ligase I in a PCNA-dependent manner. We conclude that DNA ligase I displays different influences on the two multipotent DNA polymerases delta and epsilon through PCNA. This might be of importance in the selective involvement in DNA transactions such as DNA replication and various mechanisms of DNA repair.

DNA↗
PubMed1998

Newly synthesized L-enantiomers of 3'-fluoro-modified beta-2'-deoxyribonucleoside 5'-triphosphates inhibit hepatitis B DNA polymerases but not the five cellular DNA polymerases alpha, beta, gamma, delta, and epsilon nor HIV-1 reverse transcriptase.

Novel beta-L-2',3'-dideoxy-3'-fluoro nucleosides were synthesized and further converted to their 5'-triphosphates. Their inhibitory activities against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) DNA polymerases, human immunodeficiency virus (HIV) reverse transcriptase (RT), and the cellular DNA polymerases alpha, beta, gamma, delta, and epsilon were investigated and compared with those of the corresponding 3'-fluoro-modified beta-d-analogues. The 5'-triphosphates of 3'-deoxy-3'-fluoro-beta-L-thymidine (beta-L-FTTP), 2',3'-dideoxy-3'-fluoro-beta-L-cytidine (beta-L-FdCTP), and 2',3'-dideoxy-3'-fluoro-beta-l-5-methylcytidine (beta-L-FMetdCTP) emerged as effective inhibitors of HBV/DHBV DNA polymerases (IC50 = 0.25-10.4 microM). They were either equally (FTTP) or less (FMetdCTP, FdCTP) effective than their beta-d-counterparts. Also the 5'-triphosphate of beta-L-thymidine (beta-L-TTP) was shown to be a strong inhibitor of these two viral enzymes (IC50 = 0.46/1.0 microM). However, all beta-L-FdNTPs (also beta-L-TTP) were inactive against HIV-RT, a result which contrasts sharply with the high efficiency of the beta-D- FdNTPs against this polymerase. Between the cellular DNA polymerases only the beta and gamma enzymes displayed a critical susceptibility to beta-D-FdNTPs which is largely abolished by the beta-L-enantiomers. These results recommend beta-L-FTdR, beta-L-FCdR, and beta-L-FMetCdR for further evaluation as selective inhibitors of HBV replication at the cellular level.

Animals↗
PubMed1998

Clamping down on clamps and clamp loaders--the eukaryotic replication factor C.

DNA transactions such as DNA replication and DNA repair require the concerted action of many enzymes, together with other proteins and non-protein cofactors. Among them three main accessory proteins, replication factor C (RF-C), proliferating-cell nuclear antigen (PCNA) and replication protein A (RP-A), are essential for accurate and processive DNA synthesis by DNA polymerases. RF-C is a complex consisting of five polypeptides with distinct functions. RF-C can bind to a template-primer junction and, in the presence of ATP, load the PCNA clamp onto DNA, thereby recruiting DNA polymerases to the site of DNA synthesis. RF-C not only acts as a clamp loader in DNA replication and DNA repair, but there is some evidence that it could be involved in several other processes such as transcription, S-phase checkpoint regulation, apoptosis, differentiation and telomere-length regulation.

Animals↗
PubMed1998

Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases.

The reverse transcriptase (RT) of HIV-1 and feline immunodeficiency virus (FIV) consist of two subunits of 51 kDa (p51) and 66 kDa (p66). In order to elucidate the role of p51 in the heterodimer, chimeric HIV-1/FIV RT heterodimers were constructed and characterized. The FIV RT p51/HIV-1 RT p66 chimera showed a 2.5-fold higher RNase H activity than the natural HIV-1 RT, a 50% lower strand displacement DNA synthesis activity and resistance to the two RT inhibitors 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and Nevirapine. The HIV-1 RT p51/FIV RT p66 chimera on the other hand had very similar properties to the natural FIV RT. The differences observed upon exchange of the p51 subunits suggest that the three-dimensional structure of the p51 subunit in the RT heterodimers is not completely conserved between the human and the feline lentiviruses. Finally, our data suggest an important role for the p51 subunit in maintaining the optimal structural integrity of the RT heterodimer. The different effects of the small subunits on the sensitivity to known RT inhibitors might be of importance in the development of novel drugs against HIV-1 RT.

Antiviral Agents↗
PubMed1998

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.

Amino Acid Sequence↗
PubMed1998

The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering.

The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase delta or epsilon to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.

Buffers↗
PubMed1997

Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabilizing interactions and thermodynamic or steric barriers are induced by different single amino acid substitutions.

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 (kon) and dissociation (koff) for the mutant and wild-type enzymes showed that mutations L100I and V106A increased the koff values by 12 and 8.5-fold, respectively, without significantly affecting the kon, whereas mutation K103N decreased the kon 5-fold without increasing the koff. Mutations Y181I and Y188L, on the other hand, conferred resistance to Nevirapine affecting both koff and kon 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.

Anti-HIV Agents↗
PubMed1997

Cloning, chromosomal localization, and interspecies interaction of mouse DNA polymerase delta small subunit (PolD2).

DNA polymerase delta core is a heterodimeric enzyme with a catalytic subunit of 125 kDa and a second subunit of 50 kDa with an as yet unknown function. It is an essential enzyme for DNA replication and DNA repair. We cloned the full-length cDNA encoding the DNA polymerase delta small subunit from mouse cells. The sequence of the predicted polypeptide of 51,336 Da is, like the catalytic subunit, highly conserved not only among mammals (93% identity and 96% similarity), but also between yeast and mammals (34% identity and 57% similarity). Fluorescence in situ hybridization experiments indicated that the gene for the small DNA polymerase delta of mouse is localized on chromosome 11, band A2. By using the yeast two-hybrid system we found that the mouse 125-kDa DNA polymerase catalytic subunit is able to interact with the 50-kDa subunit of the human enzyme, suggesting an in vivo interspecies interaction between the two subunits of DNA polymerase delta.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis.

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C and consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta1 and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosphorylation by CaMKII, suggesting a possible role of CaMKII in regulating the dynamics of interaction between PCNA and RF-C and thus interfering in the formation of an active sliding clamp by DNA polymerases delta and epsilon.

Animals↗
PubMed1997

Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen.

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.

Animals↗
PubMed1997

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

Animals↗
PubMed1997

Proliferating cell nuclear antigen: more than a clamp for DNA polymerases.

DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases delta and epsilon. It now appears that proliferating cell nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post-replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells.

Animals↗
PubMed1997

DNA polymerase delta, an essential enzyme for DNA transactions.

Many DNA transactions, such as replication, repair and recombination involve DNA synthesis and consequently require the action of DNA synthesizing enzymes called DNA polymerases (Pol). Eukaryotic cells contain at least six different Pols, named alpha, beta, gamma, delta, epsilon, and zeta. Among them Pol delta occupies important roles in DNA replication, nucleotide excision repair, base excision repair and VDJ recombination. Pol a has been extremely conserved in evolution from yeast to man. The function of Pol delta must be considered in the context of two other factors, called proliferating cell nuclear antigen and replication factor C, two protein complexes that build together the moving platform for Pol delta. This moving platform provides an important framework for dynamic properties of an accurate Pol delta such as its recruitment when its function is needed, the facilitation of Pol delta binding to the primer terminus, the increase in Pol delta processivity, the prevention of non-productive binding of the Pol delta to single-stranded DNA, the release of Pol delta after DNA synthesis and the bridging of Pol delta interactions to other replication proteins. In this review we summarize the current knowledge of Pol delta and will focus in particular to its structural conservation, its functional tasks in the cell and its interactions with other proteins.

Amino Acid Sequence↗
PubMed1996

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.

Amino Acid Sequence↗
PubMed1996

Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmon resonance.

The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system. Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.

Amino Acid Sequence↗