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Biomedical subjects

Vilhelm A Bohr

Publications and source records attributed to Vilhelm A Bohr.

At least 19 recordsLinked to original sources
PubMed2025

Cockayne syndrome mice reflect human kidney disease and are defective in de novo NAD biosynthesis.

Cockayne Syndrome (CS) is a premature aging disorder caused by mutations in the CSA and CSB genes involved in DNA metabolism and other cellular processes. CS patients display many features including premature aging, neurodegeneration, and kidney abnormalities. Nicotinamide dinucleotide (NAD+) deprivation has been observed in CS patient-derived cells. NAD+ has essential roles in regulating cellular health, stress responses, and renal homeostasis. While kidney dysfunction is a common feature in CS patients, its molecular pathogenesis is not understood. Here, we report that severe kidney pathology is present in CS A and B mice. We find that the NAD+ biosynthetic pathways are impaired in kidneys from these mice. Using human renal tubular epithelial cells, we show that CSA/B downregulation causes persistent activation of the ATF3 transcription factor on the quinolinate phosphoribosyl transferase gene locus, a rate-limiting enzyme in de novo NAD+ biosynthesis in the kidney, causing impaired transcription and deficient NAD+ homeostasis.

Animals↗
PubMed2006

WRN exonuclease activity is blocked by DNA termini harboring 3' obstructive groups.

Reactive oxygen species, generated either by cellular respiration or upon exposure to environmental agents such as ionizing radiation (IR), attack DNA to form a variety of oxidized base and sugar modifications. Accumulation of oxidative DNA damage has been associated with age-related disease as well as the aging process. Single-strand breaks harboring oxidative 3' obstructive termini, e.g. 3' phosphates and 3' phosphoglycolates, must be removed prior to DNA repair synthesis or ligation. In addition, 3' tyrosyl-linked protein damage, resulting from therapeutic agents such as camptothecin (CPT), must be processed to initiate repair. Several nucleases participate in DNA repair and the excision of 3' obstructive ends. As the protein defective in the segmental progeroid Werner syndrome (WRN) possesses 3'-5' exonuclease activity, and Werner syndrome cells are hypersensitive to IR and CPT, we examined for WRN exonuclease activity on 3' blocking lesions. Moreover, we compared side-by-side the activity of four prominent human 3'-5' exonucleases (WRN, APE1, TREX1, and p53) on substrates containing 3' phosphates, phosphoglycolates, and tyrosyl residues. Our studies reveal that while WRN degrades 3' hydroxyl containing substrates in a non-processive manner, it does not excise 3' phosphate, phosphoglycolate, or tyrosyl groups. In addition, we found that APE1 was most active at excising 3' blocking termini in comparison to the disease-related exonucleases TREX1, WRN, and p53 under identical physiological reaction conditions, and that TREX1 was the most powerful 3'-5' exonuclease on undamaged oligonucleotide substrates.

Cells, Cultured↗
PubMed2006

Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest.

Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, we use fluorescence resonance energy transfer (FRET) and immune-precipitation experiments to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. We verify the WRN-RAD51 and WRN-RAD54B direct interaction in vitro. Our data support a role for WRN also in the recombination step of ICL repair.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

The role of WRN in DNA repair is affected by post-translational modifications.

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by genomic instability. WRN gene encodes one of the RecQ helicase family proteins, WRN, which has ATPase, helicase, exonuclease and single stranded DNA annealing activities. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in DNA repair, replication and recombination. The role of WRN in these pathways can be modulated by its post-translational modifications in response to DNA damage. Here, we review the functional consequences of post-translational modifications on WRN as well as specific DNA repair pathways where WRN is involved and discuss how these modifications affect DNA repair pathways.

Animals↗
PubMed2006

The mechanics of base excision repair, and its relationship to aging and disease.

Base excision repair (BER) is the major pathway responsible for averting the mutagenic and cytotoxic effects of spontaneous hydrolytic, oxidative, and non-enzymatic alkylation DNA damage. In particular, this pathway recognizes and repairs base modifications, such as uracil and 8-hydroxyguanine, as well as abasic sites and DNA single-strand breaks. In this review, we outline the basic mechanics of the BER process, and describe the potential association of this pathway with aging and age-related disease, namely cancer and neurodegeneration.

Aging↗
PubMed2006

A role for WRN in telomere-based DNA damage responses.

Telomeres cap the ends of eukaryotic chromosomes and prevent them from being recognized as DNA breaks. We have shown that certain DNA damage responses induced during senescence and, at times of telomere uncapping, also can be induced by treatment of cells with small DNA oligonucleotides homologous to the telomere 3' single-strand overhang (T-oligos), implicating this overhang in generation of these telomere-based damage responses. Here, we show that T-oligo-treated fibroblasts contain gammaH2AX foci and that these foci colocalize with telomeres. T-oligos with nuclease-resistant 3' ends are inactive, suggesting that a nuclease initiates T-oligo responses. We therefore examined WRN, a 3'-->5' exonuclease and helicase mutated in Werner syndrome, a disorder characterized by aberrant telomere maintenance, premature aging, chromosomal rearrangements, and predisposition to malignancy. Normal fibroblasts and U20S osteosarcoma cells rendered deficient in WRN showed reduced phosphorylation of p53 and histone H2AX in response to T-oligo treatment. Together, these data demonstrate a role for WRN in processing of telomeric DNA and subsequent activation of DNA damage responses. The T-oligo model helps define the role of WRN in telomere maintenance and initiation of DNA damage responses after telomere disruption.

Cells, Cultured↗
PubMed2006

WRN's tenth anniversary.

Werner syndrome (WS) is a segmental progeroid syndrome in which patients display pleiotropic features of aging seen in the normal population. The advent of positional cloning in the 1990s markedly accelerated the identification of human disease-causing genes. In 1996, mutations in WRN, which was shown to encode a new, putative member of the family of RecQ DNA helicases, were identified in four patients as the cause of WS. Ten years after the identification of WRN, what have we learned about its role in WS, and its contribution to normal aging?

Adult↗
PubMed2006

[DNA repair--a fundamental factor in ageing and development of cancer].

Advanced age and increased incidence of many illnesses such as cancer are closely linked. The reasons for such a link are numerous but one important factor is DNA repair. DNA repair pathways in both nuclei and mitochondria ensure that genomic instability is minimised, thus preventing transformation and premature cellular decay. However, overall cellular DNA repair capacity decreases with age; moreover, some individuals are born with defects in repair systems. The resulting lower capacity for repair of DNA damage increases mutation load and changes normal cellular functions such as transcription, thereby contributing to the ageing process as well to the onset of various cancers. DNA repair capacity is an important cellular marker that should be considered as a standard clinical test.

Age Factors↗
PubMed2006

Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer.

Werner syndrome (WS) is an inherited disorder characterized by premature onset of aging, genomic instability, and increased cancer incidence. The disease is caused by loss of function mutations of the WRN gene, a RecQ family member with both helicase and exonuclease activities. However, despite its putative tumor-suppressor function, little is known about the contribution of WRN to human sporadic malignancies. Here, we report that WRN function is abrogated in human cancer cells by transcriptional silencing associated with CpG island-promoter hypermethylation. We also show that, at the biochemical and cellular levels, the epigenetic inactivation of WRN leads to the loss of WRN-associated exonuclease activity and increased chromosomal instability and apoptosis induced by topoisomerase inhibitors. The described phenotype is reversed by the use of a DNA-demethylating agent or by the reintroduction of WRN into cancer cells displaying methylation-dependent silencing of WRN. Furthermore, the restoration of WRN expression induces tumor-suppressor-like features, such as reduced colony formation density and inhibition of tumor growth in nude mouse xenograft models. Screening a large collection of human primary tumors (n = 630) from different cell types revealed that WRN CpG island hypermethylation was a common event in epithelial and mesenchymal tumorigenesis. Most importantly, WRN hypermethylation in colorectal tumors was a predictor of good clinical response to the camptothecin analogue irinotecan, a topoisomerase inhibitor commonly used in the clinical setting for the treatment of this tumor type. These findings highlight the importance of WRN epigenetic inactivation in human cancer, leading to enhanced chromosomal instability and hypersensitivity to chemotherapeutic drugs.

Aging, Premature↗
PubMed2006

Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links.

Cells deficient in the Werner syndrome protein (WRN) or BRCA1 are hypersensitive to DNA interstrand cross-links (ICLs), whose repair requires nucleotide excision repair (NER) and homologous recombination (HR). However, the roles of WRN and BRCA1 in the repair of DNA ICLs are not understood and the molecular mechanisms of ICL repair at the processing stage have not yet been established. This study demonstrates that WRN helicase activity, but not exonuclease activity, is required to process DNA ICLs in cells and that WRN cooperates with BRCA1 in the cellular response to DNA ICLs. BRCA1 interacts directly with WRN and stimulates WRN helicase and exonuclease activities in vitro. The interaction between WRN and BRCA1 increases in cells treated with DNA cross-linking agents. WRN binding to BRCA1 was mapped to BRCA1 452-1079 amino acids. The BRCA1/BARD1 complex also associates with WRN in vivo and stimulates WRN helicase activity on forked and Holliday junction substrates. These findings suggest that WRN and BRCA1 act in a coordinated manner to facilitate repair of DNA ICLs.

BRCA1 Protein↗
PubMed2006

TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons.

Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.

Astrocytes↗
PubMed2006

Cellular responses to DNA damage: current state of the field and review of the 52nd Benzon Symposium.

The response of cells to DNA damage is critical for maintaining genomic integrity and for preventing cancer development. Current advances in our understanding of the response of cells to DNA double-strand breaks and to stalled DNA replication forks and the relationship of these responses to human disease were discussed at the 52nd Benzon Symposium in Denmark, Copenhagen. Here we review the novel findings that were presented at this Symposium and the current state of the field.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

The Werner syndrome protein operates in base excision repair and cooperates with DNA polymerase beta.

Genome instability is a characteristic of cancer and aging, and is a hallmark of the premature aging disorder Werner syndrome (WS). Evidence suggests that the Werner syndrome protein (WRN) contributes to the maintenance of genome integrity through its involvement in DNA repair. In particular, biochemical evidence indicates a role for WRN in base excision repair (BER). We have previously reported that WRN helicase activity stimulates DNA polymerase beta (pol beta) strand displacement synthesis in vitro. In this report we demonstrate that WRN exonuclease activity can act cooperatively with pol beta, a polymerase lacking 3'-5' proofreading activity. Furthermore, using small interference RNA technology, we demonstrate that WRN knockdown cells are hypersensitive to the alkylating agent methyl methanesulfonate, which creates DNA damage that is primarily repaired by the BER pathway. In addition, repair assays using whole cell extracts from WRN knockdown cells indicate a defect in long patch (LP) BER. These findings demonstrate that WRN plays a direct role in the repair of methylation-induced DNA damage, and suggest a role for both WRN helicase and exonuclease activities together with pol beta during LP BER.

Alkylating Agents↗
PubMed2006

Cockayne syndrome group B protein has novel strand annealing and exchange activities.

Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by UV sensitivity, severe neurological abnormalities and prageroid symptoms. The CS complementation group B (CSB) protein is involved in UV-induced transcription coupled repair (TCR), base excision repair and general transcription. CSB also has a DNA-dependent ATPase activity that may play a role in remodeling chromatin in vivo. This study reports the novel finding that CSB catalyzes the annealing of complementary single-stranded DNA (ssDNA) molecules with high efficiency, and has strand exchange activity. The rate of CSB-catalyzed annealing of complementary ssDNA is 25-fold faster than the rate of spontaneous ssDNA annealing under identical in vitro conditions and the reaction occurs with a high specificity in the presence of excess non-homologous ssDNA. The specificity and intrinsic nature of the reaction is also confirmed by the observation that it is stimulated by dephosphorylation of CSB, which occurs after UV-induced DNA damage, and is inhibited in the presence of ATPgammaS. Potential roles of CSB in cooperation with strand annealing and exchange activities for TCR and homologous recombination are discussed.

Adenosine Triphosphate↗
PubMed2006

Telomere repeat binding factor 2 interacts with base excision repair proteins and stimulates DNA synthesis by DNA polymerase beta.

The ends of linear chromosomes are capped and protected by protein-DNA complexes termed telomeres. Consequences of telomere dysfunction include genomic instability that can contribute to neoplastic transformation and progression. Telomere binding proteins interact with numerous proteins involved in DNA repair, underscoring the importance of regulating DNA repair pathways at telomeres. Telomeric DNA is particularly susceptible to oxidative damage, and such damage is repaired primarily via the base excision repair (BER) pathway. Using a screen for potential interactions between telomere repeat binding factor 2 (TRF2) and proteins involved in BER of oxidized bases in vitro, we found that TRF2 physically bound DNA polymerase beta (Pol beta) and flap endonuclease 1 (FEN-1). The interactions with endogenous proteins in human cell extracts were confirmed by coimmunoprecipitation experiments. The primary binding sites for both Pol beta and FEN-1 mapped to the TRF2 NH2-terminal and COOH-terminal domains. We further tested the ability of TRF2 to modulate BER protein partners individually on a variety of substrates in vitro. TRF2 stimulated Pol beta primer extension DNA synthesis on telomeric and nontelomeric primer/template substrates, resulting in up to a 75% increase in the proportion of longer products. TRF2 also stimulated Pol beta strand displacement DNA synthesis in reconstituted BER reactions and increased the percent of long-patch BER intermediates on both telomeric and nontelomeric substrates. Potential roles of TRF2 in cooperation with BER proteins for DNA repair pathways at telomeres, as well as other genomic regions, are discussed.

DNA Damage↗
PubMed2006

Enzymatic mechanism of the WRN helicase/nuclease.

Werner syndrome (WS) is a premature aging disorder characterized by genomic instability and increased cancer risk (Martin, 1978). The WRN gene product defective in WS belongs to the RecQ family of DNA helicases (Yu et al., 1996). Mutations in RecQ family members BLM and RecQ4 result in two other disorders associated with elevated chromosomal instability and cancer, Bloom syndrome and Rothmund-Thomson syndrome, respectively (for review see Opresko et al., 2004a). RecQ helicase mutants display defects in DNA replication, recombination, and repair, suggesting a role for RecQ helicases in maintaining genomic integrity. The WRN gene encodes a 1,432 amino acid protein that has several catalytic activities (Brosh and Bohr, 2002) (Fig. 1). WRN is a DNA-dependent ATPase and utilizes the energy from ATP hydrolysis to unwind double-stranded DNA. WRN is also a 3' to 5' exonuclease, consistent with the presence of three conserved exonuclease motifs homologous to the exonuclease domain of Escherichia coli DNA polymerase I and RNase D. Most recently, WRN (Machwe et al., 2005) and other human RecQ helicases (Garcia et al., 2004; Machwe et al., 2005; Sharma et al., 2005) have been reported to possess an intrinsic single-strand annealing activity. In addition to its catalytic activities, WRN interacts with a number of proteins involved in various aspects of DNA metabolism. To understand the role of WRN in the maintenance of genome stability, a number of laboratories have undertaken a thorough characterization of its molecular and cellular functions. Here, we describe methods and approaches used for the functional and mechanistic analysis of WRN helicase or exonuclease activity. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding or exonuclease activity of WRN protein are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of WRN or related helicases or nucleases, which would serve as a platform for further investigation of its molecular and cellular functions.

Adenosine Triphosphatases↗
PubMed2006

No association between telomere length and survival among the elderly and oldest old.

BACKGROUND: The consistent findings of a negative correlation between telomere length and replicative potential of cultured cells, as well as a decreasing telomere length in a number of different tissues in humans with age, have led to the suggestion that telomeres play a role in cellular aging in vivo and ultimately even in organismal aging. Furthermore, one small longitudinal study of elderly individuals has suggested that longer telomeres are associated with better survival. METHODS: Telomere length was measured as mean terminal restriction fragment length on blood cells from 812 persons, age 73 to 101 years, who participated in population-based surveys in 1997-1998. Among the participants were 652 twins. The participants were followed up through the Danish Civil Registration system until January 2005, at which time 412 (51%) were dead. RESULTS: Univariate Cox regression analyses revealed that longer telomeres were associated with better survival (hazard ratios = 0.89 [95% confidence interval = 0.76-1.04] per 1 kb in males and 0.79 [0.72-0.88] per 1 kb in females, respectively). However, including age in the analyses changed the estimates to 0.97 (0.83-1.14) and 0.93 (0.85-1.03), respectively. Intrapair comparison showed that among 175 twin pairs in which at least one died during follow up, it was the twin with the shorter telomere length who died first in 97 (55%) of the pairs (95% confidence interval = 48-63%). We could not confirm the recently reported negative correlation between telomere length and obesity or between telomere length and smoking. CONCLUSION: This longitudinal study of the elderly and oldest old does not support the hypothesis that telomere length is a predictor for remaining lifespan once age is controlled for.

Aged↗