10 results on '"Anna Szambowska"'
Search Results
2. Cdc45-induced loading of human RPA onto single-stranded DNA
- Author
-
Piotr Prus, Frank Grosse, Ingrid Tessmer, Bernhard Schlott, Anna Szambowska, and Helmut Pospiech
- Subjects
Models, Molecular ,0301 basic medicine ,DNA, Single-Stranded ,Cell Cycle Proteins ,Genome Integrity, Repair and Replication ,dna ,Primosome ,environment and public health ,complex mixtures ,Single-stranded binding protein ,03 medical and health sciences ,0302 clinical medicine ,dna replication fork ,repair and replication ,genome integrity ,Replication Protein A ,Genetics ,Humans ,antibodies ,Replication protein A ,carrier proteins ,Binding Sites ,DNA clamp ,oligonucleotides ,biology ,DNA replication ,mechlorethamine ,single-stranded ,DNA Replication Fork ,Molecular biology ,nucleotides ,stabilization ,trypsin ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,biology.protein ,Biophysics ,Replisome ,Primase ,new mexico ,dna helicases ,030217 neurology & neurosurgery ,cell cycle proteins ,complex ,surface plasmon resonance ,Protein Binding - Abstract
Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8–10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork.
- Published
- 2017
3. S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci
- Author
-
Helmut Pospiech, Florian Kraft, Alexandra Zielinski, Anja Weise, Kerstin Borgmann, Bernhard Schlott, Kristin Mrasek, Franziska Foertsch, Anna Szambowska, Christian Melle, and Frank Grosse
- Subjects
0301 basic medicine ,DNA Repair ,DNA repair ,Cell Survival ,RAD51 ,Down-Regulation ,Biology ,Cell Line ,Homology directed repair ,03 medical and health sciences ,0302 clinical medicine ,Report ,Humans ,Homologous Recombination ,Molecular Biology ,Replication protein A ,Chromosome Aberrations ,Genome, Human ,S100 Proteins ,Cell Biology ,DNA repair protein XRCC4 ,Molecular biology ,Non-homologous end joining ,030104 developmental biology ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques ,DNA mismatch repair ,Calcium ,Mutant Proteins ,Rad51 Recombinase ,Developmental Biology ,Nucleotide excision repair ,DNA Damage ,Protein Binding - Abstract
The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca2+, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.
- Published
- 2016
4. Assembly of the Cdc45-MCM2-7-GINS Complex, the Replication Helicase
- Author
-
Helmut Pospiech and Anna Szambowska
- Subjects
CMG complex ,Control of chromosome duplication ,DNA replication initiation ,Minichromosome maintenance ,Chemistry ,Origin recognition complex ,Pre-replication complex ,GINS ,dnaB helicase ,Cell biology - Abstract
In eukaryotes, a crucial step during the initiation of DNA replication is the timely formation and activation of the replicative DNA helicase composed of Cdc45, MCM2-7 and GINS (CMG). The dynamic and spatio-temporal events leading to the ordered and stepwise assembly of the CMG helicase are tightly regulated. Multiple assembly factors ensure in this way that replication occurs only once per cell cycle. The MCM2-7 helicase is loaded in an inactive form onto double-stranded DNA in the G1 phase of the cell cycle, whereas the fully reconstituted CMG complex is assembled and positioned onto single-stranded DNA during the S phase. Thus, DNA plays an important and active role in these events. In this chapter we summarize and discuss our current knowledge about the appropriate recruitment and assembly of the CMG complex into the active eukaryotic replicative DNA helicase, emphasizing the crucial role of DNA in this process. We finally outline how the number of active CMG complexes formed is restricted during unperturbed DNA synthesis.
- Published
- 2016
5. Modulation of λ plasmid and phage DNA replication by Escherichia coli SeqA protein
- Author
-
Sylwia Barańska, Alicja Węgrzyn, Grzegorz Węgrzyn, Monika Glinkowska, Magdalena Narajczyk, and Anna Szambowska
- Subjects
DNA Replication ,Escherichia coli Proteins ,DNA replication ,Replication Origin ,Biology ,Bacteriophage lambda ,Microbiology ,Molecular biology ,DNA-Binding Proteins ,Viral Proteins ,Replication factor C ,Plasmid ,SeqA protein domain ,Control of chromosome duplication ,Replication Initiation ,DNA, Viral ,Mutation ,Escherichia coli ,Origin recognition complex ,Replicon ,Bacterial Outer Membrane Proteins ,Plasmids - Abstract
SeqA protein, a main negative regulator of the replication initiation of the Escherichia coli chromosome, also has several other functions which are still poorly understood. It was demonstrated previously that in seqA mutants the copy number of another replicon, the lambda plasmid, is decreased, and that the activity of the lambda p(R) promoter (whose function is required for stimulation of ori lambda) is lower than that in the wild-type host. Here, SeqA-mediated regulation of lambda phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on ori lambda-dependent DNA replication in vitro was observed, indicating that a direct regulation of lambda DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled lambda DNA was monitored after phage infection of host cells, strongly suggested the early appearance of sigma replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of lambda plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable lambda replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the lambda O protein was easily detectable, indicating that such a heritable complex can function in the mutant. To investigate the influence of seqA gene function on heritable complex- and transcription-dependent lambda DNA replication, the efficiency of lambda plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of lambda plasmid replication. These results indicate that unlike oriC, SeqA modulates lambda DNA replication indirectly, most probably by influencing the stability of the lambda replication complex and the transcriptional activation of ori lambda.
- Published
- 2007
6. The C-terminal domain of the Escherichia coli RNA polymerase α subunit plays a role in the CI-dependent activation of the bacteriophage λ pM promoter
- Author
-
Stephen J. W. Busby, Anna Szambowska, Barbara Kędzierska, Mark S. Thomas, Anna Herman-Antosiewicz, David J. Lee, and Grzegorz Wegrzyn
- Subjects
Gene Expression Regulation, Viral ,Transcriptional Activation ,Protein subunit ,Repressor ,Biology ,DNA-binding protein ,chemistry.chemical_compound ,Viral Proteins ,Transcription (biology) ,RNA polymerase ,Genetics ,Viral Regulatory and Accessory Proteins ,Promoter Regions, Genetic ,Molecular Biology ,Models, Genetic ,Activator (genetics) ,C-terminus ,Escherichia coli Proteins ,DNA-Directed RNA Polymerases ,Molecular biology ,Bacteriophage lambda ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Repressor Proteins ,chemistry ,Amino Acid Substitution ,DNA - Abstract
The bacteriophage lambda p(M) promoter is required for maintenance of the lambda prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the lambda repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of p(M). CI activates p(M) through cooperative binding to the O(R)1 and O(R)2 sites within the O(R) operator, with the O(R)2-bound CI dimer making contact with domain 4 of the RNA polymerase sigma subunit (sigma(4)). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of p(M). We also show that the location of alphaCTD at the p(M) promoter changes in the presence of CI. Thus, in the absence of CI, one alphaCTD is located on the DNA at position -44 relative to the transcription start site, whereas in the presence of CI, alphaCTD is located at position -54, between the CI-binding sites at O(R)1 and O(R)2. These results suggest that contacts between CI and both alphaCTD and sigma are required for efficient CI-dependent activation of p(M).
- Published
- 2007
7. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex
- Author
-
Christoph Wiedemann, Sabine Häfner, Anna Szambowska, Oliver Ohlenschläger, Matthias Görlach, and Karl-Heinz Gührs
- Subjects
Models, Molecular ,Methanobacteriaceae ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Archaeal Proteins ,Recombinant Fusion Proteins ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Winged Helix ,adenosine triphosphatases ,eukaryotic cell ,dna helicases ,chimera organism ,dna ,archaea ,sulfolobus ,helix (snails) ,Protein Structure, Secondary ,Adenosine Triphosphate ,Minichromosome maintenance ,Structural Biology ,Genetics ,Amino Acid Sequence ,Peptide sequence ,Phylogeny ,Adenosine Triphosphatases ,Minichromosome Maintenance Proteins ,Sequence Homology, Amino Acid ,biology ,ved/biology ,Hydrolysis ,Sulfolobus solfataricus ,DNA Helicases ,Helicase ,biology.organism_classification ,Protein Structure, Tertiary ,Sulfolobus ,Biochemistry ,Mutation ,Helix ,biology.protein ,Linker - Abstract
The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ?wings? of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short ?-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short ?-helical linker element and by N-terminal residues of the first ?-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.
- Published
- 2015
8. A dual promoter system regulating λ DNA replication initiation
- Author
-
Sylwia Barańska, Anna Szambowska, Monika Glinkowska, Grzegorz Węgrzyn, Magdalena Narajczyk, and Paweł Olszewski
- Subjects
DNA Replication ,DNA replication initiation ,Transcription, Genetic ,Eukaryotic DNA replication ,Replication Origin ,Biology ,Genome Integrity, Repair and Replication ,Pre-replication complex ,Molecular biology ,Bacteriophage lambda ,DNA replication factor CDT1 ,Viral Proteins ,Replication factor C ,Licensing factor ,Control of chromosome duplication ,DNA, Viral ,Mutation ,Genetics ,biology.protein ,Origin recognition complex ,Promoter Regions, Genetic ,Plasmids - Abstract
Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes.
- Published
- 2014
9. DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding
- Author
-
Christian Usskilat, Anna Szambowska, Helmut Pospiech, Frank Grosse, Piotr Prus, Petri Kursula, and Ingrid Tessmer
- Subjects
Protein Folding ,DNA polymerase ,DNA, Single-Stranded ,Cell Cycle Proteins ,Genome Integrity, Repair and Replication ,DNA polymerase delta ,Protein Structure, Secondary ,Genetics ,Humans ,ddc:610 ,Replication protein A ,chemistry.chemical_classification ,DNA ligase ,DNA clamp ,biology ,Circular bacterial chromosome ,DNA replication ,DNA ,Molecular biology ,DNA-Binding Proteins ,chemistry ,biology.protein ,Biophysics ,Nucleic Acid Conformation ,DNA supercoil ,Protein Binding - Abstract
The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3′ protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3′–5′ polarity and, thereby acts as a molecular ‘wedge’ to initiate DNA strand displacement.
- Published
- 2014
10. Influence of the Escherichia coli oxyR gene function on lambda prophage maintenance
- Author
-
Monika Glinkowska, Marcin Łoś, Anna Szambowska, Joanna Całkiewicz, Joanna M. Łoś, Grzegorz Węgrzyn, Agata Czyż, Alicja Węgrzyn, Borys Wróbel, and Anna Herman-Antosiewicz
- Subjects
Gene Expression Regulation, Viral ,Prophages ,Mutant ,Molecular Sequence Data ,Repressor ,Biology ,medicine.disease_cause ,Biochemistry ,Microbiology ,Shiga toxin-encoding lambdoid phages ,Lysogen ,Genetics ,medicine ,Escherichia coli ,λ Prophage induction ,OxyR protein ,SOS response ,Promoter Regions, Genetic ,SOS Response, Genetics ,Molecular Biology ,Prophage ,Original Paper ,Binding Sites ,Base Sequence ,Escherichia coli Proteins ,Promoter ,General Medicine ,Ci protein ,Hydrogen Peroxide ,Molecular biology ,Bacteriophage lambda ,Repressor Proteins ,Oxidative Stress ,bacteria ,Virus Activation - Abstract
In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of lambda prophage. Here, we demonstrate that H2O2-mediated lambda prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a lambda lysogen in the presence of H2O2. On the other hand, stimulation of the p(M) promoter by cI857 overproduced from a multicopy plasmid was decreased in the DeltaoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p(M) promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p(M) promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced lambda prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating lambda prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.
- Published
- 2010
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.