Publications by authors named "Barbara van Loon"

37 Publications

Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities.

Cell Rep Med 2021 Apr 8;2(4):100240. Epub 2021 Apr 8.

Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway.

Essential E3 ubiquitin ligase HUWE1 (HECT, UBA, and WWE domain containing 1) regulates key factors, such as p53. Although mutations in cause heterogenous neurodevelopmental X-linked intellectual disabilities (XLIDs), the disease mechanisms common to these syndromes remain unknown. In this work, we identify p53 signaling as the central process altered in HUWE1-promoted XLID syndromes. By focusing on Juberg-Marsidi syndrome (JMS), one of the severest XLIDs, we show that increased p53 signaling results from p53 accumulation caused by HUWE1 p.G4310R destabilization. This further alters cell-cycle progression and proliferation in JMS cells. Modeling of JMS neurodevelopment reveals majorly impaired neural differentiation accompanied by increased p53 signaling. The neural differentiation defects can be successfully rescued by reducing p53 levels and restoring the expression of p53 target genes, in particular . In summary, our findings suggest that increased p53 signaling underlies HUWE1-promoted syndromes and impairs XLID JMS neural differentiation.
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http://dx.doi.org/10.1016/j.xcrm.2021.100240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080178PMC
April 2021

Loss of Mediator complex subunit 13 (MED13) promotes resistance to alkylation through cyclin D1 upregulation.

Nucleic Acids Res 2021 02;49(3):1470-1484

Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, 7049 Trondheim, Norway.

Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.
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http://dx.doi.org/10.1093/nar/gkaa1289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897519PMC
February 2021

Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression.

Nat Commun 2019 11 29;10(1):5460. Epub 2019 Nov 29.

Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology (NTNU), 7491, Trondheim, Norway.

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases, including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here, we show that AAG binds to chromatin and forms complex with RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes, aberrantly methylated bases accumulate towards the 3'end in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide insights into genome stability maintenance in actively transcribing chromatin and reveal roles of aberrantly methylated bases in regulation of gene expression.
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http://dx.doi.org/10.1038/s41467-019-13394-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884549PMC
November 2019

Characterization of FGD5 Expression in Primary Breast Cancers and Lymph Node Metastases.

J Histochem Cytochem 2018 11 27;66(11):787-799. Epub 2018 Jul 27.

Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway.

Faciogenital dysplasia 5 ( FGD5) amplification drives tumor cell proliferation, and is present in 9.5% of breast cancers. We describe FGD5 expression, assess associations between FGD5 amplification and FGD5 expression, and assess FGD5 expression in relation to proliferation and prognosis. FGD5 immunohistochemistry was done on primary tumors ( n=829) and lymph node metastases ( n=231) from a cohort of Norwegian patients. We explored associations between FGD5 amplification, FGD5 expression, and proliferation, and analyzed the prognostic value of FGD5 expression by estimating cumulative risks of death and hazard ratios (HRs). We identified nuclear and cytoplasmic expression in 64% and 73% of primary tumors, respectively, and found an association between gene amplification and nuclear expression ( p=0.02). The proportion of cases with FGD5 expression was higher in lymph node metastases, compared with primary tumors ( p=0.004 for nuclear and p=0.001 for cytoplasmic staining). Neither proliferation nor prognosis was associated with FGD5 expression (age-adjusted HR 1.12 [95% confidence interval = 0.89-1.41] for nuclear expression; and 0.88 [95% CI = 0.70-1.12] for cytoplasmic expression). FGD5 is expressed in a high proportion of breast cancers and lymph node metastases. There was a correlation between FGD5 amplification and nuclear expression, but no association between FGD5 expression and proliferation or prognosis.
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http://dx.doi.org/10.1369/0022155418792032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213565PMC
November 2018

Publisher Correction: Impaired oxidative stress response characterizes HUWE1-promoted X-linked intellectual disability.

Sci Rep 2018 Apr 12;8(1):6010. Epub 2018 Apr 12.

Department of Molecular Mechanisms of Disease, University of Zurich, Zürich, 8057, Switzerland.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-24189-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897351PMC
April 2018

HUWE1 variants cause dominant X-linked intellectual disability: a clinical study of 21 patients.

Eur J Hum Genet 2018 01 27;26(1):64-74. Epub 2017 Nov 27.

Genomics Institute, MultiCare Health System, Tacoma, WA, USA.

Whole-gene duplications and missense variants in the HUWE1 gene (NM_031407.6) have been reported in association with intellectual disability (ID). Increased gene dosage has been observed in males with non-syndromic mild to moderate ID with speech delay. Missense variants reported previously appear to be associated with severe ID in males and mild or no ID in obligate carrier females. Here, we report the largest cohort of patients with HUWE1 variants, consisting of 14 females and 7 males, with 15 different missense variants and one splice site variant. Clinical assessment identified common clinical features consisting of moderate to profound ID, delayed or absent speech, short stature with small hands and feet and facial dysmorphism consisting of a broad nasal tip, deep set eyes, epicanthic folds, short palpebral fissures, and a short philtrum. We describe for the first time that females can be severely affected, despite preferential inactivation of the affected X chromosome. Three females with the c.329 G  >  A p.Arg110Gln variant, present with a phenotype of mild ID, specific facial features, scoliosis and craniosynostosis, as reported previously in a single patient. In these females, the X inactivation pattern appeared skewed in favour of the affected transcript. In summary, HUWE1 missense variants may cause syndromic ID in both males and females.
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http://dx.doi.org/10.1038/s41431-017-0038-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788272PMC
January 2018

Impaired oxidative stress response characterizes HUWE1-promoted X-linked intellectual disability.

Sci Rep 2017 11 8;7(1):15050. Epub 2017 Nov 8.

Department of Molecular Mechanisms of Disease, University of Zurich, Zürich, 8057, Switzerland.

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID.
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http://dx.doi.org/10.1038/s41598-017-15380-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678168PMC
November 2017

Living on the Edge: DNA Polymerase Lambda between Genome Stability and Mutagenesis.

Chem Res Toxicol 2017 11 8;30(11):1936-1941. Epub 2017 Sep 8.

DNA Enzymology & Molecular Virology and Cell Nucleus & DNA replication Units, Institute of Molecular Genetics IGM-CNR , via Abbiategrasso 207, I-27100 Pavia, Italy.

In human cells, only four DNA polymerases (pols) are necessary and sufficient for the duplication of the genetic information. However, more than a dozen DNA pols are required to maintain its integrity. Such a high degree of specialization makes DNA repair pols able to cope with specific lesions or repair pathways. On the other hand, the same DNA pols can have partially overlapping roles, which could result in possible conflicts of functions, if the DNA pols are not properly regulated. DNA pol λ is a typical example of such an enzyme. It is a multifunctional enzyme, endowed with special structural and biochemical properties, which make it capable of participating in different DNA repair pathways such as base excision repair, nonhomologous end joining, and translesion synthesis. However, when mutated or deregulated, DNA pol λ can also be a source of genetic instability. Its multiple roles in DNA damage tolerance and its ability in promoting tumor progression make it also a possible target for novel anticancer approaches.
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http://dx.doi.org/10.1021/acs.chemrestox.7b00152DOI Listing
November 2017

Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions.

Blood 2017 09 21;130(13):1523-1534. Epub 2017 Aug 21.

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for , a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of , essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.
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http://dx.doi.org/10.1182/blood-2017-01-764274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620415PMC
September 2017

Ribonucleotide incorporation by human DNA polymerase η impacts translesion synthesis and RNase H2 activity.

Nucleic Acids Res 2017 03;45(5):2600-2614

DNA Enzymology & Molecular Virology and Cell Nucleus & DNA replication Units, Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.
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http://dx.doi.org/10.1093/nar/gkw1275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389505PMC
March 2017

Bypass of Mutagenic O(6)-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases.

Chem Res Toxicol 2016 09 24;29(9):1493-503. Epub 2016 Aug 24.

Department of Health Sciences and Technology, ETH Zürich , Schmelzbergstrasse 9, 8092 Zürich, Switzerland.

The generation of chemical alkylating agents from nitrosation of glycine and bile acid conjugates in the gastrointestinal tract is hypothesized to initiate carcinogenesis. O(6)-carboxymethylguanine (O(6)-CMG) is a product of DNA alkylation derived from nitrosated glycine. Although the tendency of the structurally related adduct O(6)-methylguanine to code for the misincoporation of TTP during DNA replication is well-established, the impact of the presence of the O(6)-CMG adduct in a DNA template on the efficiency and fidelity of translesion DNA synthesis (TLS) by human DNA polymerases (Pols) has hitherto not been described. Herein, we characterize the ability of the four human TLS Pols η, ι, κ, and ζ and the replicative Pol δ to bypass O(6)-CMG in a prevalent mutational hot-spot for colon cancer. The results indicate that Pol η replicates past O(6)-CMG, incorporating dCMP or dAMP, whereas Pol κ incorporates dCMP only, and Pol ι incorporates primarily dTMP. Additionally, the subsequent extension step was carried out with high efficiency by TLS Pols η, κ, and ζ, while Pol ι was unable to extend from a terminal mismatch. These results provide a first basis of O(6)-CMG-promoted base misincorporation by Y- and B-family polymerases potentially leading to mutational signatures associated with colon cancer.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00168DOI Listing
September 2016

HUWE1 mutations in Juberg-Marsidi and Brooks syndromes: the results of an X-chromosome exome sequencing study.

BMJ Open 2016 Apr 29;6(4):e009537. Epub 2016 Apr 29.

Greenwood Genetic Center, Greenwood, South Carolina, USA.

Background: X linked intellectual disability (XLID) syndromes account for a substantial number of males with ID. Much progress has been made in identifying the genetic cause in many of the syndromes described 20-40 years ago. Next generation sequencing (NGS) has contributed to the rapid discovery of XLID genes and identifying novel mutations in known XLID genes for many of these syndromes.

Methods: 2 NGS approaches were employed to identify mutations in X linked genes in families with XLID disorders. 1 involved exome sequencing of genes on the X chromosome using the Agilent SureSelect Human X Chromosome Kit. The second approach was to conduct targeted NGS sequencing of 90 known XLID genes.

Results: We identified the same mutation, a c.12928 G>C transversion in the HUWE1 gene, which gives rise to a p.G4310R missense mutation in 2 XLID disorders: Juberg-Marsidi syndrome (JMS) and Brooks syndrome. Although the original families with these disorders were considered separate entities, they indeed overlap clinically. A third family was also found to have a novel HUWE1 mutation.

Conclusions: As we identified a HUWE1 mutation in an affected male from the original family reported by Juberg and Marsidi, it is evident the syndrome does not result from a mutation in ATRX as reported in the literature. Additionally, our data indicate that JMS and Brooks syndromes are allelic having the same HUWE1 mutation.
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http://dx.doi.org/10.1136/bmjopen-2015-009537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854010PMC
April 2016

Impact of ribonucleotide incorporation by DNA polymerases β and λ on oxidative base excision repair.

Nat Commun 2016 Feb 26;7:10805. Epub 2016 Feb 26.

DNA Enzymology &Molecular Virology Unit, Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions.
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http://dx.doi.org/10.1038/ncomms10805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773436PMC
February 2016

The interaction between ALKBH2 DNA repair enzyme and PCNA is direct, mediated by the hydrophobic pocket of PCNA and perturbed in naturally-occurring ALKBH2 variants.

DNA Repair (Amst) 2015 Nov 14;35:13-8. Epub 2015 Sep 14.

Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. Electronic address:

Human AlkB homolog 2 (ALKBH2) is a DNA repair enzyme that catalyzes the direct reversal of DNA methylation damage through oxidative demethylation. While ALKBH2 colocalizes with proliferating cell nuclear antigen (PCNA) in DNA replication foci, it remains unknown whether these two proteins alone form a complex or require additional components for interaction. Here, we demonstrate that ALKBH2 can directly interact with PCNA independent from other cellular factors, and we identify the hydrophobic pocket of PCNA as the key domain mediating this interaction. Moreover, we find that PCNA association with ALKBH2 increases significantly during DNA replication, suggesting that ALKBH2 forms a cell-cycle dependent complex with PCNA. Intriguingly, we show that an ALKBH2 germline variant, as well as a variant found in cancer, display altered interaction with PCNA. Our studies reveal the ALKBH2 binding interface of PCNA and indicate that both germline and somatic ALKBH2 variants could have cellular effects on ALKBH2 function in DNA repair.
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http://dx.doi.org/10.1016/j.dnarep.2015.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206904PMC
November 2015

DNA polymerases: Biology, diseases and biomedical applications.

DNA Repair (Amst) 2015 May;29:1-3

Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

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http://dx.doi.org/10.1016/j.dnarep.2015.04.001DOI Listing
May 2015

Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase η by reducing synthesis in the presence of the major cisplatin DNA adduct.

J Am Chem Soc 2015 Apr 6;137(14):4728-34. Epub 2015 Apr 6.

§Department of Health Sciences and Technology, ETH Zürich, Schmelzbergstrasse 9, 8092 Zürich, Switzerland.

Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template.
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http://dx.doi.org/10.1021/ja512547gDOI Listing
April 2015

Agnoprotein of polyomavirus BK interacts with proliferating cell nuclear antigen and inhibits DNA replication.

Virol J 2015 Feb 1;12. Epub 2015 Feb 1.

UiT - The Arctic University of Norway, Faculty of Health Sciences, Department of Medical Biology, Molecular Inflammation Research Group, Tromsø NO-9037, Norway.

Background: The human polyomavirus BK expresses a 66 amino-acid peptide referred to as agnoprotein. Though mutants lacking agnoprotein are severely reduced in producing infectious virions, the exact function of this peptide remains incompletely understood. To elucidate the function of agnoprotein, we searched for novel cellular interaction partners.

Methods: Yeast-two hybrid assay was performed with agnoprotein as bait against human kidney and thymus libraries. The interaction between agnoprotein and putative partners was further examined by GST pull down, co-immunoprecipitation, and fluorescence resonance energy transfer studies. Biochemical and biological studies were performed to examine the functional implication of the interaction of agnoprotein with cellular target proteins.

Results: Proliferating cell nuclear antigen (PCNA), which acts as a processivity factor for DNA polymerase δ, was identified as an interaction partner. The interaction between agnoprotein and PCNA is direct and occurs also in human cells. Agnoprotein exerts an inhibitory effect on PCNA-dependent DNA synthesis in vitro and reduces cell proliferation when ectopically expressed. Overexpression of PCNA restores agnoprotein-mediated inhibition of cell proliferation.

Conclusion: Our data suggest that PCNA is a genuine interaction partner of agnoprotein and the inhibitory effect on PCNA-dependent DNA synthesis by the agnoprotein may play a role in switching off (viral) DNA replication late in the viral replication cycle when assembly of replicated genomes and synthesized viral capsid proteins occurs.
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http://dx.doi.org/10.1186/s12985-014-0220-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318453PMC
February 2015

Pharmacological Inhibition of poly(ADP-ribose) polymerases improves fitness and mitochondrial function in skeletal muscle.

Cell Metab 2014 Jun 8;19(6):1034-41. Epub 2014 May 8.

Laboratory for Integrative and Systems Physiology, École Polytechnique Fédérale de Lausanne, Station 15, CH-1015 Lausanne, Switzerland. Electronic address:

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.
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http://dx.doi.org/10.1016/j.cmet.2014.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047186PMC
June 2014

DNA damage response and DNA repair - dog as a model?

BMC Cancer 2014 Mar 19;14:203. Epub 2014 Mar 19.

Division of Radiation Oncology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.

Background: Companion animals like dogs frequently develop tumors with age and similarly to human malignancies, display interpatient tumoral heterogeneity. Tumors are frequently characterized with regard to their mutation spectra, changes in gene expression or protein levels. Among others, these changes affect proteins involved in the DNA damage response (DDR), which served as a basis for the development of numerous clinically relevant cancer therapies. Even though the effects of different DNA damaging agents, as well as DDR kinetics, have been well characterized in mammalian cells in vitro, very little is so far known about the kinetics of DDR in tumor and normal tissues in vivo.

Discussion: Due to (i) the similarities between human and canine genomes, (ii) the course of spontaneous tumor development, as well as (iii) common exposure to environmental agents, canine tumors are potentially an excellent model to study DDR in vivo. This is further supported by the fact that dogs show approximately the same rate of tumor development with age as humans. Though similarities between human and dog osteosarcoma, as well as mammary tumors have been well established, only few studies using canine tumor samples addressed the importance of affected DDR pathways in tumor progression, thus leaving many questions unanswered.

Summary: Studies in humans showed that misregulated DDR pathways play an important role during tumor development, as well as in treatment response. Since dogs are proposed to be a good tumor model in many aspects of cancer research, we herein critically investigate the current knowledge of canine DDR and discuss (i) its future potential for studies on the in vivo level, as well as (ii) its possible translation to veterinary and human medicine.
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http://dx.doi.org/10.1186/1471-2407-14-203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995094PMC
March 2014

DNA polymerase δ-interacting protein 2 is a processivity factor for DNA polymerase λ during 8-oxo-7,8-dihydroguanine bypass.

Proc Natl Acad Sci U S A 2013 Nov 4;110(47):18850-5. Epub 2013 Nov 4.

Institute of Molecular Genetics-Consiglio Nazionale delle Ricerche, I-27100 Pavia, Italy.

The bypass of DNA lesions by the replication fork requires a switch between the replicative DNA polymerase (Pol) and a more specialized translesion synthesis (TLS) Pol to overcome the obstacle. DNA Pol δ-interacting protein 2 (PolDIP2) has been found to physically interact with Pol η, Pol ζ, and Rev1, suggesting a possible role of PolDIP2 in the TLS reaction. However, the consequences of PolDIP2 interaction on the properties of TLS Pols remain unknown. Here, we analyzed the effects of PolDIP2 on normal and TLS by five different human specialized Pols from three families: Pol δ (family B), Pol η and Pol ι (family Y), and Pol λ and Pol β (family X). Our results show that PolDIP2 also physically interacts with Pol λ, which is involved in the correct bypass of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions. This interaction increases both the processivity and catalytic efficiency of the error-free bypass of a 8-oxo-G lesion by both Pols η and λ, but not by Pols β or ι. Additionally, we provide evidence that PolDIP2 stimulates Pol δ without affecting its fidelity, facilitating the switch from Pol δ to Pol λ during 8-oxo-G TLS. PolDIP2 stimulates Pols λ and η mediated bypass of other common DNA lesions, such as abasic sites and cyclobutane thymine dimers. Finally, PolDIP2 silencing increases cell sensitivity to oxidative stress and its effect is further potentiated in a Pol λ deficient background, suggesting that PolDIP2 is an important mediator for TLS.
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http://dx.doi.org/10.1073/pnas.1308760110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839753PMC
November 2013

Handling the 3-methylcytosine lesion by six human DNA polymerases members of the B-, X- and Y-families.

Nucleic Acids Res 2014 Jan 4;42(1):553-66. Epub 2013 Oct 4.

Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.

Alkylating agents often generate 3-methylcytosine (3meC) lesions that are efficiently repaired by AlkB homologues. If AlkB homologue proteins are not functional, or the number of 3meC lesions exceeds the cellular repair capacity, the damage will persist in the genome and become substrate of DNA polymerases (Pols). Though alkylating agents are present in our environment and used in the clinics, currently nothing is known about the impact of 3meC on the accuracy and efficiency of human Pols. Here we compared the 3meC bypass properties of six human Pols belonging to the three families: B (Pol δ), X (Pols β and λ) and Y (Pols κ, ι and η). We show that under replicative conditions 3meC impairs B-family, blocks X-family, but not Y-family Pols, in particular Pols η and ι. These Pols successfully synthesize opposite 3meC; Pol ι preferentially misincorporates dTTP and Pol η dATP. The most efficient extenders from 3meC base-paired primers are Pols κ and η. Finally, using xeroderma pigmentosum variant patient cell extracts, we provide evidence that the presence of functional Pol η is mandatory to efficiently overcome 3meC by mediating complete bypass or extension. Our data suggest that Pol η is crucial for efficient 3meC bypass.
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http://dx.doi.org/10.1093/nar/gkt889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874200PMC
January 2014

Genotype-phenotype analysis of S326C OGG1 polymorphism: a risk factor for oxidative pathologies.

Free Radic Biol Med 2013 Oct 28;63:401-9. Epub 2013 May 28.

Department of Environment and Primary Prevention, Istituto Superiore di Sanità, 00161 Rome, Italy.

8-Oxoguanine DNA glycosylase (OGG) activity was measured by an in vitro assay in lymphocytes of healthy volunteers genotyped for various OGG1 polymorphisms. Only homozygous carriers of the polymorphic C326 allele showed a significantly lower OGG activity compared to the homozygous S326 genotype. The purified S326C OGG1 showed a decreased ability to complete the repair synthesis step in a base excision repair reaction reconstituted in vitro. The propensity of this variant to dimerize as well as its catalytic impairment were shown to be enhanced under oxidizing conditions. Mass spectrometry revealed that the extra cysteine of the variant protein is involved in disulfide bonds compatible with significant conformational changes and/or dimerization. We propose that the S326C OGG1 catalytic impairment and its susceptibility to dimerization and disulfide bond formation in an oxidizing environment all concur to decrease repair capacity. Consequently, the C326 homozygous carriers may be at increased risk of oxidative pathologies.
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http://dx.doi.org/10.1016/j.freeradbiomed.2013.05.031DOI Listing
October 2013

The human Tim-Tipin complex interacts directly with DNA polymerase epsilon and stimulates its synthetic activity.

J Biol Chem 2013 May 19;288(18):12742-52. Epub 2013 Mar 19.

Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.

The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ε. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ε directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ε bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork.
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http://dx.doi.org/10.1074/jbc.M112.398073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642320PMC
May 2013

Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB).

DNA Repair (Amst) 2013 Mar 3;12(3):177-87. Epub 2013 Jan 3.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair.
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http://dx.doi.org/10.1016/j.dnarep.2012.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998512PMC
March 2013

Base excision repair in physiology and pathology of the central nervous system.

Int J Mol Sci 2012 Nov 30;13(12):16172-222. Epub 2012 Nov 30.

Institute for Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, 8057 Zürich, Switzerland.

Relatively low levels of antioxidant enzymes and high oxygen metabolism result in formation of numerous oxidized DNA lesions in the tissues of the central nervous system. Accumulation of damage in the DNA, due to continuous genotoxic stress, has been linked to both aging and the development of various neurodegenerative disorders. Different DNA repair pathways have evolved to successfully act on damaged DNA and prevent genomic instability. The predominant and essential DNA repair pathway for the removal of small DNA base lesions is base excision repair (BER). In this review we will discuss the current knowledge on the involvement of BER proteins in the maintenance of genetic stability in different brain regions and how changes in the levels of these proteins contribute to aging and the onset of neurodegenerative disorders.
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http://dx.doi.org/10.3390/ijms131216172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546685PMC
November 2012

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint.

Nucleic Acids Res 2013 Jan 30;41(1):229-41. Epub 2012 Oct 30.

Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.
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http://dx.doi.org/10.1093/nar/gks1016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592438PMC
January 2013

Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage.

Nucleic Acids Res 2012 Sep 29;40(17):8449-59. Epub 2012 Jun 29.

Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

Reactive oxygen species constantly generated as by-products of cellular metabolism readily attack genomic DNA creating mutagenic lesions such as 7,8-dihydro-8-oxo-guanine (8-oxo-G) that promote aging. 8-oxo-G:A mispairs arising during DNA replication are eliminated by base excision repair initiated by the MutY DNA glycosylase homologue (MUTYH). Here, by using formaldehyde crosslinking in mammalian cell extracts, we demonstrate that the WRN helicase/exonuclease defective in the premature aging disorder Werner syndrome (WS) is recruited to DNA duplex containing an 8-oxo-G:A mispair in a manner dependent on DNA polymerase λ (Polλ) that catalyzes accurate DNA synthesis over 8-oxo-G. Similarly, by immunofluorescence, we show that Polλ is required for accumulation of WRN at sites of 8-oxo-G lesions in human cells. Moreover, we show that nuclear focus formation of WRN and Polλ induced by oxidative stress is dependent on ongoing DNA replication and on the presence of MUTYH. Cell viability assays reveal that depletion of MUTYH suppresses the hypersensitivity of cells lacking WRN and/or Polλ to oxidative stress. Biochemical studies demonstrate that WRN binds to the catalytic domain of Polλ and specifically stimulates DNA gap filling by Polλ over 8-oxo-G followed by strand displacement synthesis. Our results suggest that WRN promotes long-patch DNA repair synthesis by Polλ during MUTYH-initiated repair of 8-oxo-G:A mispairs.
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http://dx.doi.org/10.1093/nar/gks648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458577PMC
September 2012

Regulation of oxidative DNA damage repair: the adenine:8-oxo-guanine problem.

Cell Cycle 2012 Mar 15;11(6):1070-5. Epub 2012 Mar 15.

Institute for Veterinary Biochemistry and Molecular Biology; University of Zürich-Irchel; Zürich, Switzerland.

Reactive oxygen species (ROS) constantly attack DNA. One of the best-characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). Many human diseases, such as cancer and neurodegenerative disorders, have been correlated with oxidative DNA damage. In the last few years, DNA polymerase (Pol) λ, one of the 15 cellular Pols, has been identified to play an important role in performing accurate translesion synthesis over 8-oxo-G. This is eminently important, since normally faithful replicative Pols α, δ and ε, with their tight active center, often wrongly incorporate adenine (A) opposite the 8-oxo-G lesion. A:8- oxo-G mispairs are accurately repaired by the pathway identified in our laboratory involving MutY DNA glycosylase homolog (MutYH) and Pol λ. Until now, very little was known about the spatial and temporal regulation of Pol λ and MutYH in active repair complexes. We now showed in our latest publication that the E3 ligase Mule can ubiquitinate and degrade Pol λ, and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. In contrast, phosphorylation of Pol λ by Cdk2/cyclinA counteracts this degradation by recruiting it to MutYH on chromatin to form active 8-oxo-G repair complexes.
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http://dx.doi.org/10.4161/cc.11.6.19448DOI Listing
March 2012

Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination.

Proc Natl Acad Sci U S A 2012 Jan 27;109(2):437-42. Epub 2011 Dec 27.

Institute for Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and β, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ's affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation.
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http://dx.doi.org/10.1073/pnas.1110449109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258598PMC
January 2012

Ubiquitylation of DNA polymerase λ.

FEBS Lett 2011 Sep 8;585(18):2826-30. Epub 2011 Apr 8.

Institute for Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, 8057 Zürich, Switzerland.

DNA polymerase (pol) λ, one of the 15 cellular pols, belongs to the X family. It is a small 575 amino-acid protein containing a polymerase, a dRP-lyase, a proline/serine rich and a BRCT domain. Pol λ shows various enzymatic activities including DNA polymerization, terminal transferase and dRP-lyase. It has been implicated to play a role in several DNA repair pathways, particularly base excision repair (BER), non-homologous end-joining (NHEJ) and translesion DNA synthesis (TLS). Similarly to other DNA repair enzymes, pol λ undergoes posttranslational modifications during the cell cycle that regulate its stability and possibly its subcellular localization. Here we describe our knowledge about ubiquitylation of pol λ and the impact of this modification on its regulation.
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http://dx.doi.org/10.1016/j.febslet.2011.03.069DOI Listing
September 2011