Search PubMed⌕ Search

Biomedical subjects

W Vermeulen

Publications and source records attributed to W Vermeulen.

At least 19 recordsLinked to original sources
PubMed2006

Interaction of nucleotide excision repair factors RPA and XPA with DNA containing bulky photoreactive groups imitating damages.

Interaction of nucleotide excision repair factors--replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)--with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged nucleotides was investigated. Efficiency of photoaffinity modification of two proteins by photoreactive DNAs varied depending on DNA structure and type of photoreactive group. The secondary structure of DNA and, first of all, the presence of extended single-stranded parts plays a key role in recognition by RPA. However, it was shown that RPA efficiently interacts with DNA duplex containing a bulky substituent at the 5 -end of a nick. XPA was shown to prefer the nicked DNA; however, this protein was cross-linked with approximately equal efficiency by single-stranded and double-stranded DNA containing a bulky substituent inside the strand. XPA seems to be sensitive not only to the structure of DNA double helix, but also to a bulky group incorporated into DNA. The mechanism of damage recognition in the process of nucleotide excision repair is discussed.

Base Sequence↗
PubMed2004

Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants.

All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c(2), the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome c(2) and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.

Cytochromes c2↗
PubMed2002

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

DNA↗
PubMed2001

Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts.

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.

3T3 Cells↗
PubMed2001

Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching.

Regulation and structural requirements of vital nuclear processes such as DNA replication, transcription, RNA processing and DNA repair inside the eukaryote nucleus are as yet poorly understood. Although a wealth of evidence exists pointing to a considerable degree of spatial organisation of chromatin and nuclear processes, there are still questions concerning the dynamics and interaction of nuclear proteins that remain unanswered. The cloning of the gene encoding the green fluorescent protein (GFP) has revolutionised the study of proteins in living cells. The expression of recombinant cDNA fusion plasmids of GFP and proteins of interest currently enables the investigation of those proteins in living cells. Time-lapse confocal microscopy as well as quantitative fluorescence methods such as fluorescence redistribution after photobleaching (FRAP) and fluorescence resonance energy transfer are widely applied to living cells expressing GFP fusion proteins. This review gives an overview of the current state of knowledge of nuclear structure and function. In particular, the different applications of FRAP technology to study the dynamics of GFP-tagged nuclear proteins will be summarised.

Cell Nucleus↗
PubMed2001

Sequential assembly of the nucleotide excision repair factors in vivo.

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

Cell Line↗
PubMed2001

A temperature-sensitive disorder in basal transcription and DNA repair in humans.

The xeroderma pigmentosum group D (XPD) helicase subunit of TFIIH functions in DNA repair and transcription initiation. Different mutations in XPD give rise to three ultraviolet-sensitive syndromes: the skin cancer-prone disorder xeroderma pigmentosum (XP), in which repair of ultraviolet damage is affected; and the severe neurodevelopmental conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD). In the latter two, the basal transcription function of TFIIH is also presumed to be affected. Here we report four unusual TTD patients with fever-dependent reversible deterioration of TTD features such as brittle hair. Cells from these patients show an in vivo temperature-sensitive defect of transcription and DNA repair due to thermo-instability of TFIIH. Our findings reveal the clinical consequences of impaired basal transcription and mutations in very fundamental processes in humans, which previously were only known in lower organisms.

Base Sequence↗
PubMed2000

Cloning of a human homolog of the yeast nucleotide excision repair gene MMS19 and interaction with transcription repair factor TFIIH via the XPB and XPD helicases.

Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro but have an undefined role in vivo and may act at the interface of NER and other cellular processes. An intriguing example is the Saccharomyces cerevisiae Mms19 protein that has an unknown dual function in NER and RNA polymerase II transcription. Here we report the cloning and characterization of a human homolog, designated hMMS19, that encodes a 1030 amino acid protein with 26% identity and 51% similarity to S.cerevisiae Mms19p and with a strikingly similar size. The expression profile and nuclear location are consistent with a repair function. Co-immunoprecipitation experiments revealed that hMMS19 directly interacts with the XPB and XPD subunits of NER-transcription factor TFIIH. These findings extend the conservation of the NER apparatus and the link between NER and basal transcription and suggest that hMMS19 exerts its function in repair and transcription by interacting with the XPB and XPD helicases.

Amino Acid Sequence↗
PubMed2000

XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

Amino Acid Motifs↗
PubMed2000

TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair.

TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5' nor 3' incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients.

Adenosine Triphosphatases↗
PubMed2000

Sublimiting concentration of TFIIH transcription/DNA repair factor causes TTD-A trichothiodystrophy disorder.

The repair-deficient form of trichothiodystrophy (TTD) most often results from mutations in the genes XPB or XPD, encoding helicases of the transcription/repair factor TFIIH. The genetic defect in a third group, TTD-A, is unknown, but is also caused by dysfunctioning TFIIH. None of the TFIIH subunits carry a mutation and TFIIH from TTD-A cells is active in both transcription and repair. Instead, immunoblot and immunofluorescence analyses reveal a strong reduction in the TFIIH concentration. Thus, the phenotype of TTD-A appears to result from sublimiting amounts of TFIIH, probably due to a mutation in a gene determining the complex stability. The reduction of TFIIH mainly affects its repair function and hardly influences transcription.

Abnormalities, Multiple↗
PubMed2000

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

Adenosine Triphosphate↗
PubMed1999

Action of DNA repair endonuclease ERCC1/XPF in living cells.

To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility was monitored in living Chinese hamster ovary cells. In the absence of DNA damage, the complex moved freely through the nucleus, with a diffusion coefficient (15 +/- 5 square micrometers per second) consistent with its molecular size. Ultraviolet light-induced DNA damage caused a transient dose-dependent immobilization of ERCC1/XPF, likely due to engagement of the complex in a single repair event. After 4 minutes, the complex regained mobility. These results suggest (i) that NER operates by assembly of individual NER factors at sites of DNA damage rather than by preassembly of holocomplexes and (ii) that ERCC1/XPF participates in repair of DNA damage in a distributive fashion rather than by processive scanning of large genome segments.

Animals↗
PubMed1998

Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein.

Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.

Adenosine Triphosphatases↗
PubMed1998

Affinity purification of human DNA repair/transcription factor TFIIH using epitope-tagged xeroderma pigmentosum B protein.

TFIIH is a high molecular weight complex with a remarkable dual function in nucleotide excision repair and initiation of RNA polymerase II transcription. Mutations in the largest subunits, the XPB and XPD helicases, are associated with three inherited disorders: xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy. To facilitate the purification and biochemical characterization of this intricate complex, we generated a cell line stably expressing tagged XPB, allowing the immunopurification of the XPB protein and associated factors. Addition of two tags, a N-terminal hexameric histidine stretch and a C-terminal hemagglutinin epitope, to this highly conserved protein did not interfere with its functioning in repair and transcription. The hemagglutinin epitope allowed efficient TFIIH immunopurification to homogeneity from a fractionated whole cell extract in essentially one step. We conclude that the predominant active form of TFIIH is composed of nine subunits and that there is one molecule of XPB per TFIIH complex. The affinity-purified complex exhibits all expected TFIIH activities: DNA-dependent ATPase, helicase, C-terminal domain kinase, and participation in in vitro and in vivo nucleotide excision repair and in vitro transcription. The affinity purification procedure described here is fast and simple, does not require extensive chromatographic procedures, and yields highly purified, active TFIIH.

Blotting, Western↗
PubMed1997

Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP.

A connection between transcription and DNA repair was demonstrated previously through the characterization of TFIIH. Using filter binding as well as in vitro transcription challenge competition assays, we now show that the promoter recognition factor TATA box-binding protein (TBP)/TFIID binds selectively to and is sequestered by cisplatin- or UV-damaged DNA, either alone or in the context of a larger protein complex including TFIIH. Computer-assisted 3D structural analysis reveals a remarkable similarity between the structure of the TATA box as found in its TBP complex and that of either platinated or UV-damaged oligonucleotides. Thus, cisplatin-treated or UV-irradiated DNA could be used as a competing binding site which may lure TBP/TFIID away from its normal promoter sequence, partially explaining the phenomenon of DNA damage-induced inhibition of RNA synthesis. Consistent with an involvement of damaged DNA-specific binding of TBP in inhibiting transcription, we find that microinjection of additional TBP in living human fibroblasts alleviates the reduction in RNA synthesis after UV irradiation. Future anticancer drugs could be designed with the consideration of lesion recognition by TBP and their ability to reduce transcription.

Cisplatin↗