DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficie... more DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase. The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E. coli is thought to target and block DnaB-dependent DNA replication. The toxicity of Kid is antagonized through interaction with the Kis antidote protein and the resultant complex can then act as a transcriptional regulator for the parD system. Crystals of selenomethionine-incorporated Kid have been obtained by the hanging-drop vapour-diffusion method using potassium phosphate as the precipitant. The crystals belong to the monoclinic system, space group P2(1), have unit-cell parameters a = 32.9, b = 45.0, c = 64.4 A, beta = 96.2 degrees and diffract to a d(min) of better than 1.8 A on a synchrotron-radiation source. The determination of the structure of Kid will permit a better understanding of its interactions with DnaB and Kis and allow the evolutionary relations...
Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The res... more Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The resulting mutant retains significant catalytic activity upon heating above 60 °C (and up to 100 °C) and cooling down, whereas wild-type lipase precipitates irreversibly and does not show significant activity in these conditions. Kinetic thermostability of proteins has not been characterized well on the molecular structure level so far, therefore our aim is to study it using NMR spectroscopy. Here, nearly complete (1)H, (13)C and (15)N resonance assignments are reported for wild-type and mutant Lipase A. Chemical shifts were used to predict secondary structure elements of both Lipase A variants.
Numerous proteins are involved in the multiple pathways of the DNA damage response network and pl... more Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.
Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. ... more Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. Histone chaperones are regulators of this process by preventing formation of non-nucleosomal histone-DNA complexes. Aprataxin and polynucleotide kinase like factor (APLF) is a non-homologous endjoining (NHEJ) DNA repair factor that possesses histone chaperone activity in its acidic domain (APLF AD). Here, we studied the molecular basis of this activity using biochemical and structural methods. We find that APLF AD is intrinsically disordered and binds histone complexes (H3-H4) 2 and H2A-H2B specifically and with high affinity. APLF AD prevents unspecific complex formation between H2A-H2B and DNA in a chaperone assay, establishing for the first time its specific histone chaperone function for H2A-H2B. On the basis of a series of nuclear magnetic resonance studies, supported by mutational analysis, we show that the APLF AD histone binding domain uses two aromatic side chains to anchor to the ␣1-␣2 patches on both H2A and H2B, thereby covering most of their DNA-interaction surface. An additional binding site on both APLF AD and H2A-H2B may be involved in the handoff between APLF and DNA or other chaperones. Together, our data support the view that APLF provides not only a scaffold but also generic histone chaperone activity for the NHEJ-complex.
Multitechnique characterization of six technical lignins including nuclear magnetic resonance and... more Multitechnique characterization of six technical lignins including nuclear magnetic resonance and size exclusion chromatography studies.
The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function ... more The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the Nucleotide Excision Repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wildtype and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface where, in contrast to F231, L231 lacks interactions stabilizing the XPF-ERCC1 complex. One of the two anchor points is severely distorted and this results in a more dynamic complex causing reduced stability and an increased dissociation rate of the mutant complex as compared to wildtype...
NMR experiments showed close conformational similarity of thermally treated LipA variant XI to th... more NMR experiments showed close conformational similarity of thermally treated LipA variant XI to the native protein. Thermal inactivation profiles were recorded for further characterization of the recovered protein. The profiles revealed significant differences (Fig. ). Importantly, protein subjected to thermal denaturation and re-folding at either 60 or 80 °C retains more activity after the heating and cooling process than native variant XI. To prove that indeed active site residues are responsible for the activity retention of variant XI, control experiments were carried out using appropriate LipA mutants, specifically those having mutations at the active site residues (S77C, D133N and H156L). An expression clone of variant XI cloned into pET-22b (+) was obtained in a previous study S1 . This construct was subjected to site-directed mutagenesis to obtain S77C, D133N and H156L mutants. PCR and DNA preparation/sequencing were performed as described for construct preparation (see Methods in the main text).
Of the enolates tested, this one binds the strongest to 1. This is likely due to the rigid struct... more Of the enolates tested, this one binds the strongest to 1. This is likely due to the rigid structure of the enolate, which diverges the keto oxygens in an optimum manner to complement the con-vergence of the host hydrogen-bond donors of 1. In contrast, the enolates of 2,4-...
... in general, we studied this repressor as a dimer alone and in presence of ligands using high-... more ... in general, we studied this repressor as a dimer alone and in presence of ligands using high-resolution NMR in combination with SAXS and ligand ... 328, 521 – 548 (2005) 3. Wilson, CJ, Zhan, H., Swint-Kruse, L. and Matthews, KS, CMLS 64, 3 – 16, (2007) 4. Peters, F., Maestre ...
This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from collea... more This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from colleagues, many of whom have worked with him, and others who admire his work and have been stimulated by his research. The contributions show current research in biomolecular NMR, spin hyperpolarisation and spin chemistry, including CIDNP (chemically induced dynamic nuclear polarisation), topics to which he has contributed enormously. His proposal of the radical pair mechanism was the birth of the field of spin chemistry, and the laser CIDNP NMR experiment on a protein was a major breakthrough in hyperpolarisation research. He set milestones for biomolecular NMR by developing computational methods for protein structure determination, including restrained molecular dynamics and 3D NMR methodology. With a lac repressor headpiece, he determined one of the first protein structures determined by NMR. His studies of the lac repressor provided the first examples of detailed studies of protein nucleic acid complexes by NMR. This deepened our understanding of protein DNA recognition and led to a molecular model for protein sliding along the DNA. Furthermore, he played a leading role in establishing the cluster of NMR large-scale facilities in Europe. This editorial gives an introduction to the publication and is followed by a biography describing his contributions to magnetic resonance.
Protein science : a publication of the Protein Society, 2016
Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit... more Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β-sheets with each four anti-parallel strands, followed by a C-terminal α-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermo...
The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream... more The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of the DNA damage. Functional studies have provided insight into the binding of ERCC1-XPF to various DNA substrates. However, since no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the Helix-hairpin-Helix domains of XPF and ERCC-XPF and show that the binding to ss/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences, but with a clear preference for guanine containing substrates. NMR titration experiments and in vitro DNA binding assays show that also within the heterodimeric ERCC1-XPF complex XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-...
DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficie... more DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase. The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E. coli is thought to target and block DnaB-dependent DNA replication. The toxicity of Kid is antagonized through interaction with the Kis antidote protein and the resultant complex can then act as a transcriptional regulator for the parD system. Crystals of selenomethionine-incorporated Kid have been obtained by the hanging-drop vapour-diffusion method using potassium phosphate as the precipitant. The crystals belong to the monoclinic system, space group P2(1), have unit-cell parameters a = 32.9, b = 45.0, c = 64.4 A, beta = 96.2 degrees and diffract to a d(min) of better than 1.8 A on a synchrotron-radiation source. The determination of the structure of Kid will permit a better understanding of its interactions with DnaB and Kis and allow the evolutionary relations...
Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The res... more Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The resulting mutant retains significant catalytic activity upon heating above 60 °C (and up to 100 °C) and cooling down, whereas wild-type lipase precipitates irreversibly and does not show significant activity in these conditions. Kinetic thermostability of proteins has not been characterized well on the molecular structure level so far, therefore our aim is to study it using NMR spectroscopy. Here, nearly complete (1)H, (13)C and (15)N resonance assignments are reported for wild-type and mutant Lipase A. Chemical shifts were used to predict secondary structure elements of both Lipase A variants.
Numerous proteins are involved in the multiple pathways of the DNA damage response network and pl... more Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.
Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. ... more Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. Histone chaperones are regulators of this process by preventing formation of non-nucleosomal histone-DNA complexes. Aprataxin and polynucleotide kinase like factor (APLF) is a non-homologous endjoining (NHEJ) DNA repair factor that possesses histone chaperone activity in its acidic domain (APLF AD). Here, we studied the molecular basis of this activity using biochemical and structural methods. We find that APLF AD is intrinsically disordered and binds histone complexes (H3-H4) 2 and H2A-H2B specifically and with high affinity. APLF AD prevents unspecific complex formation between H2A-H2B and DNA in a chaperone assay, establishing for the first time its specific histone chaperone function for H2A-H2B. On the basis of a series of nuclear magnetic resonance studies, supported by mutational analysis, we show that the APLF AD histone binding domain uses two aromatic side chains to anchor to the ␣1-␣2 patches on both H2A and H2B, thereby covering most of their DNA-interaction surface. An additional binding site on both APLF AD and H2A-H2B may be involved in the handoff between APLF and DNA or other chaperones. Together, our data support the view that APLF provides not only a scaffold but also generic histone chaperone activity for the NHEJ-complex.
Multitechnique characterization of six technical lignins including nuclear magnetic resonance and... more Multitechnique characterization of six technical lignins including nuclear magnetic resonance and size exclusion chromatography studies.
The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function ... more The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the Nucleotide Excision Repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wildtype and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface where, in contrast to F231, L231 lacks interactions stabilizing the XPF-ERCC1 complex. One of the two anchor points is severely distorted and this results in a more dynamic complex causing reduced stability and an increased dissociation rate of the mutant complex as compared to wildtype...
NMR experiments showed close conformational similarity of thermally treated LipA variant XI to th... more NMR experiments showed close conformational similarity of thermally treated LipA variant XI to the native protein. Thermal inactivation profiles were recorded for further characterization of the recovered protein. The profiles revealed significant differences (Fig. ). Importantly, protein subjected to thermal denaturation and re-folding at either 60 or 80 °C retains more activity after the heating and cooling process than native variant XI. To prove that indeed active site residues are responsible for the activity retention of variant XI, control experiments were carried out using appropriate LipA mutants, specifically those having mutations at the active site residues (S77C, D133N and H156L). An expression clone of variant XI cloned into pET-22b (+) was obtained in a previous study S1 . This construct was subjected to site-directed mutagenesis to obtain S77C, D133N and H156L mutants. PCR and DNA preparation/sequencing were performed as described for construct preparation (see Methods in the main text).
Of the enolates tested, this one binds the strongest to 1. This is likely due to the rigid struct... more Of the enolates tested, this one binds the strongest to 1. This is likely due to the rigid structure of the enolate, which diverges the keto oxygens in an optimum manner to complement the con-vergence of the host hydrogen-bond donors of 1. In contrast, the enolates of 2,4-...
... in general, we studied this repressor as a dimer alone and in presence of ligands using high-... more ... in general, we studied this repressor as a dimer alone and in presence of ligands using high-resolution NMR in combination with SAXS and ligand ... 328, 521 – 548 (2005) 3. Wilson, CJ, Zhan, H., Swint-Kruse, L. and Matthews, KS, CMLS 64, 3 – 16, (2007) 4. Peters, F., Maestre ...
This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from collea... more This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from colleagues, many of whom have worked with him, and others who admire his work and have been stimulated by his research. The contributions show current research in biomolecular NMR, spin hyperpolarisation and spin chemistry, including CIDNP (chemically induced dynamic nuclear polarisation), topics to which he has contributed enormously. His proposal of the radical pair mechanism was the birth of the field of spin chemistry, and the laser CIDNP NMR experiment on a protein was a major breakthrough in hyperpolarisation research. He set milestones for biomolecular NMR by developing computational methods for protein structure determination, including restrained molecular dynamics and 3D NMR methodology. With a lac repressor headpiece, he determined one of the first protein structures determined by NMR. His studies of the lac repressor provided the first examples of detailed studies of protein nucleic acid complexes by NMR. This deepened our understanding of protein DNA recognition and led to a molecular model for protein sliding along the DNA. Furthermore, he played a leading role in establishing the cluster of NMR large-scale facilities in Europe. This editorial gives an introduction to the publication and is followed by a biography describing his contributions to magnetic resonance.
Protein science : a publication of the Protein Society, 2016
Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit... more Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β-sheets with each four anti-parallel strands, followed by a C-terminal α-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermo...
The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream... more The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of the DNA damage. Functional studies have provided insight into the binding of ERCC1-XPF to various DNA substrates. However, since no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the Helix-hairpin-Helix domains of XPF and ERCC-XPF and show that the binding to ss/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences, but with a clear preference for guanine containing substrates. NMR titration experiments and in vitro DNA binding assays show that also within the heterodimeric ERCC1-XPF complex XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-...
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Papers by Rolf Boelens