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Papers by Miriana Petrovich

Nucleic Acids Research, 2009

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially sing... more Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single- stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It

Journal of molecular biology, Jan 21, 2006

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain.... more The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition sta...

Proceedings of the National Academy of Sciences, 2003

The WW domains are small proteins that contain a three-stranded, antiparallel ␤-sheet. The 40-res... more The WW domains are small proteins that contain a three-stranded, antiparallel ␤-sheet. The 40-residue murine FBP28 WW domain rapidly formed twirling ribbon-like fibrils at physiological temperature and pH, with morphology typical of amyloid fibrils. These ribbons were unusually wide and well ordered, making them highly suitable for structural studies. Their x-ray and electrondiffraction patterns displayed the characteristic amyloid fiber 0.47-nm reflection of the cross-␤ diffraction signature. Both conventional and electron cryomicroscopy showed clearly that the ribbons were composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis of the fiber. There was a region of lower density along the center of each filament. Lateral association of these filaments generated twisted, often interlinked, sheets up to 40 nm wide and many microns in length. The pitch of the helix varied from 60 to 320 nm, depending on the width of the ribbon. The wild-type FBP28 fibers were formed under conditions in which multiexponential folding kinetics is observed in other studies and which was attributed to a change in the mechanism of folding. It is more likely that those phases result from initial events in the off-pathway aggregation observed here.

Proceedings of the National Academy of Sciences, 2006

Human CA150, a transcriptional activator, binds to and is codeposited with huntingtin during Hunt... more Human CA150, a transcriptional activator, binds to and is codeposited with huntingtin during Huntington's disease. The second WW domain of CA150 is a three-stranded ␤-sheet that folds in vitro in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue nonnative ␤-hairpin. The structure we report has similarities to the hairpin formed by the A protofilament, yet also contains closely packed side-chains in a ''steric zipper'' arrangement found in the cross-␤ spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.

Proceedings of the National Academy of Sciences, 2013

Proceedings of the National Academy of Sciences, 2013

Nucleic Acids Research, 2009

is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome ... more is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K d of 1.95 AE 0.19 kM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM-p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices a1 and a2 of the HMG-box constitute the main p53binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM-p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM-p53 interaction are discussed.

Journal of Molecular Biology, 2009

Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. I... more Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. Its pattern is altered in cancer and often results in the hypermethylation of the promoter regions and abnormal expression of tumour suppressor genes. Methylation of CpG dinucleotides located in the binding sites of transcription factors may contribute to the development of cancers by preventing their binding or altering their specificity. We studied the effects of CpG methylation on DNA recognition by the tumour suppressor p53, a transcription factor involved in the response to carcinogenic stress. p53 recognises a large number of DNA sequences, many of which contain CpG dinucleotides. We systematically substituted a CpG dinucleotide at each position in the consensus p53 DNA binding sequence and identified substitutions tolerated by p53. We compared the binding affinities of methylated versus non-methylated sequences by fluorescence anisotropy titration. We found that binding of p53 was not affected by cytosine methylation in a majority of cases. However, for a few sequences containing multiple CpG dinucleotides, such as sites in the RB and Met genes, methylation resulted in a four-to sixfold increase in binding of p53. This approach can be used to quantify the effects of CpG methylation on the DNA recognition by other DNA-binding proteins.

Journal of Molecular Biology, 2006

BMC Genomics, 2009

Background: Transcription factors activate their target genes by binding to specific response ele... more Background: Transcription factors activate their target genes by binding to specific response elements. Many transcription factor families evolved from a common ancestor by gene duplication and subsequent divergent evolution. Members of the p53 family, which play key roles in cell-cycle control and development, share conserved DNA binding and oligomerisation domains but exhibit distinct functions. In this study, the molecular basis of the functional divergence of related transcription factors was investigated.

Science (New York, N.Y.), Jan 4, 2011

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules ... more Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.

Nucleic Acids Research, 2009

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially sing... more Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single- stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It

Journal of molecular biology, Jan 21, 2006

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain.... more The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition sta...

Proceedings of the National Academy of Sciences, 2003

The WW domains are small proteins that contain a three-stranded, antiparallel ␤-sheet. The 40-res... more The WW domains are small proteins that contain a three-stranded, antiparallel ␤-sheet. The 40-residue murine FBP28 WW domain rapidly formed twirling ribbon-like fibrils at physiological temperature and pH, with morphology typical of amyloid fibrils. These ribbons were unusually wide and well ordered, making them highly suitable for structural studies. Their x-ray and electrondiffraction patterns displayed the characteristic amyloid fiber 0.47-nm reflection of the cross-␤ diffraction signature. Both conventional and electron cryomicroscopy showed clearly that the ribbons were composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis of the fiber. There was a region of lower density along the center of each filament. Lateral association of these filaments generated twisted, often interlinked, sheets up to 40 nm wide and many microns in length. The pitch of the helix varied from 60 to 320 nm, depending on the width of the ribbon. The wild-type FBP28 fibers were formed under conditions in which multiexponential folding kinetics is observed in other studies and which was attributed to a change in the mechanism of folding. It is more likely that those phases result from initial events in the off-pathway aggregation observed here.

Proceedings of the National Academy of Sciences, 2006

Human CA150, a transcriptional activator, binds to and is codeposited with huntingtin during Hunt... more Human CA150, a transcriptional activator, binds to and is codeposited with huntingtin during Huntington's disease. The second WW domain of CA150 is a three-stranded ␤-sheet that folds in vitro in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue nonnative ␤-hairpin. The structure we report has similarities to the hairpin formed by the A protofilament, yet also contains closely packed side-chains in a ''steric zipper'' arrangement found in the cross-␤ spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.

Proceedings of the National Academy of Sciences, 2013

Proceedings of the National Academy of Sciences, 2013

Nucleic Acids Research, 2009

is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome ... more is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K d of 1.95 AE 0.19 kM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM-p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices a1 and a2 of the HMG-box constitute the main p53binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM-p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM-p53 interaction are discussed.

Journal of Molecular Biology, 2009

Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. I... more Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. Its pattern is altered in cancer and often results in the hypermethylation of the promoter regions and abnormal expression of tumour suppressor genes. Methylation of CpG dinucleotides located in the binding sites of transcription factors may contribute to the development of cancers by preventing their binding or altering their specificity. We studied the effects of CpG methylation on DNA recognition by the tumour suppressor p53, a transcription factor involved in the response to carcinogenic stress. p53 recognises a large number of DNA sequences, many of which contain CpG dinucleotides. We systematically substituted a CpG dinucleotide at each position in the consensus p53 DNA binding sequence and identified substitutions tolerated by p53. We compared the binding affinities of methylated versus non-methylated sequences by fluorescence anisotropy titration. We found that binding of p53 was not affected by cytosine methylation in a majority of cases. However, for a few sequences containing multiple CpG dinucleotides, such as sites in the RB and Met genes, methylation resulted in a four-to sixfold increase in binding of p53. This approach can be used to quantify the effects of CpG methylation on the DNA recognition by other DNA-binding proteins.

Journal of Molecular Biology, 2006

BMC Genomics, 2009

Background: Transcription factors activate their target genes by binding to specific response ele... more Background: Transcription factors activate their target genes by binding to specific response elements. Many transcription factor families evolved from a common ancestor by gene duplication and subsequent divergent evolution. Members of the p53 family, which play key roles in cell-cycle control and development, share conserved DNA binding and oligomerisation domains but exhibit distinct functions. In this study, the molecular basis of the functional divergence of related transcription factors was investigated.

Science (New York, N.Y.), Jan 4, 2011

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules ... more Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.