Heinz Peter Nasheuer | University of Galway (original) (raw)

Papers by Heinz Peter Nasheuer

The term ‘Hallmarks of Cancer’ was coined by Hanahan and Weinberg in their influential reviews an... more The term ‘Hallmarks of Cancer’ was coined by Hanahan and Weinberg in their influential reviews and they described genome instability as a property of cells enabling cancer development [1, 2]. Accurate DNA replication of genomes is central to diminish genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase, during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exo...

Irish Journal of Earth Sciences, 2020

William King (1809-86) was the founding Professor of Mineralogy and Geology at Queen's College Ga... more William King (1809-86) was the founding Professor of Mineralogy and Geology at Queen's College Galway (QCG), one of three regional colleges opened in 1849 to provide secular university-level education in Ireland. King came from a modest background and despite lacking third-level qualifications, began publishing on palaeontological and geological matters in the 1840s. These early contributions aided his application for the professorship in Galway, particularly his seminal 1850 monograph on the Permian fossils of England, which was in preparation at the time. During his first two decades at QCG, King maintained an up-to-date teaching programme in geology and palaeontology, played a key role in establishing the natural history museum and further developed his research portfolio. He investigated several topics of international interest, including the supposed earliest fossils of living organisms and the emerging evidence for fossil humans. King's achievements were impressive, particularly as he was essentially self-taught and also considering the isolated and poor economic standing of Galway at the time. The Queen's University in Ireland (QUI) bestowed its first ever honorary Doctor of Science on William King in 1870 in recognition of his distinguished geological research, and also to mark a refocussing of the university curriculum to better reflect the importance of science. King's award came at a time when the education system was coming under increasing scrutiny in Ireland, and as part of these reforms QUI was dissolved in 1882 and replaced by the Royal University of Ireland. One of the final acts of QUI was to award a large number of former graduates with master's degrees. A select few were conferred with honorary doctorates, including King's eldest son, William Jr., who had been amongst the first students to enter QCG in 1849 and, after graduating, enjoyed a distinguished career with the Geological Survey of India. Father and son thus achieved the unique honour of being the first and last recipients of a Doctor of Science (honoris causa) from QUI for their geological endeavours.

Irish Journal of Earth Sciences, 2015

Molecular and Cellular Biology, 1991

We have investigated the DNA polymerase alpha promoter sequence requirements for the expression o... more We have investigated the DNA polymerase alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNase ...

Nucleic Acids Research, 1998

Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved b... more Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase α-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase α-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knockout mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.

Molecular and Cellular Biology, 1996

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while... more Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alp...

FEBS Journal, 2013

The replication factor Cdc45 has essential functions in the initiation and elongation steps of eu... more The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays an important role in the intra-S-phase checkpoint. Its interactions with other replication proteins during the cell cycle and after intra-S-phase checkpoint activation are only partially characterized. Here, we present that the C terminal part of Cdc45 may mediate its interactions with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A (RPA) and DNA polymerase " are maximal during S phase. Following UVC-induced DNA damage, Cdc45-Claspin complex formation is reduced whereas the binding of Cdc45 to RPA is not affected. We also show that treatment of cells with UCN-01 and Phosphatidylinisitol 3-kinase-like kinase inhibitors does not rescue the UV-induced destabilisation of Cdc45-Claspin interactions, suggesting that the loss of interaction between Cdc45 and Claspin occurs upstream of ATR activation in the intra-S-phase checkpoint.

FEBS Open Bio, 2022

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step fo... more The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that whilst Tag131-627 (V350E/P417D) and Tag131-627 (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag1-708 and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules, but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regards to Okazaki fragment initiation.

Journal of virology, 1997

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and i... more The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replica...

The EMBO Journal, 1989

Communicated by F.Eckstein DNA polymerase-primase complex, isolated with an apparently undegraded... more Communicated by F.Eckstein DNA polymerase-primase complex, isolated with an apparently undegraded a-subunit, was immunoaffinitypurified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the a-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage 4PX174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10-6 and 10 x 10-6. This value reflects the spontaneous mutation frequency of 4X174am16 phages in Escherichia coli, and is 10to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10-7 (1/2 880 000) for dGMP:Ttemp,,te mispairs, between 10-7 and 10-8 for dCMP:Ttempiate (1/35 000 000), dCMP:Atemplate (1/18 200 000) and dAMP:Gte.,a mispairs (1/16 500 000), and below 10-8 (1/100 000 000) for dTM[:Ttnpae, dGMP:Anpate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'-5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide. Upon replacing dGTP by its phosphorothioate analogue at equimolar concentrations of the four nucleoside triphosphates, a 2-fold increase in the number of revertants was observed; biasing dGTPaS 9-and 30-fold over dATP and dCTP, respectively, led to a 50-fold increase in the number of revertants. Taken together, these observations suggest that the 3'-5' exonuclease present in immunoaffinity purified human polymerase-primase proofreads nucleotide misinsertions during DNA synthesis. The exonuclease contributes at least one to two orders of magnitude to the high fidelity characteristics of the intact polymerase-primase complex.

Molecular and Cellular Biology, 1995

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human c... more Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were req...

Subcellular Biochemistry, 2010

Journal of Biological Chemistry

ABSTRACT

Cancer Research, 2014

Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is ... more Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-stranded break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phase of the cell cycle and all three subunits of RPA contain phosphorylation sites. The exact set of HR-relevant phosphorylation sites of RPA is unknown. In this study, the phosphorylation sites of chromatin-bound RPA in S and G2 phase were identified. After DNA damage, phosphorylation included pSer4/8, pSer12, pThr21, pSer23 and pSer33 of RPA2. Phosphorylation of these sites increased with time after DNA damage. Using a general ATM/ATR phosphorylation antibody, only RPA2 had substantial pSer/pThr ATM/ATR signals. Additionally, pTyr was detected on RPA1 and RPA2 and wa...

European Journal of Biochemistry, 1994

DNA-polymerase-a-primase complex contains four subunits, pl80, p68, p58, and p48, and comprises a... more DNA-polymerase-a-primase complex contains four subunits, pl80, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-polymerase-a-primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNApolymerase-a-primase. The pl80, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-polymerase-aprimase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-a-primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.

Chromosoma, 1992

The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-... more The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine

Oncogene, 1999

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein in... more Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumorsuppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase aprimase complex (pol-prim), and the cellular singlestrand DNA binding protein RPA. Highly puri®ed p53 protein bound to immobilized Tag with an apparent binding constant of 2610 8 M 71. Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4610 8 M 71 , when RPA was coupled to the sensor chip via its smallest subunit, and 1610 8 M 71 , when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase a-primase complex (pol-prim) with a K A value of 1610 10 M 71. Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2610 10 M 71 and 5610 9 M 71 , respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic eect when p53 bound to higher order complexes of polprim. A truncated form of p53, consisting of amino acids 1 ± 320, bound pol-prim by four orders of magnitude less eciently. Therefore, an intact C-terminus of p53 seems to be important for ecient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and Tag. However there was no evidence for the existence of a ternary complex consisting of Tag , pol-prim, and p53.

Nucleic Acids Research, 2009

The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N... more The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during Sand M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.