65 research outputs found
Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance
We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase Ī¹ (POLĪ¹) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved. Mutation of residues S315 and D48/D49, which abrogate p53 interactions with the DNA repair and replication proteins topoisomerase I and RPA, respectively, and residues L22/W23, which disrupt formation of p53-POLĪ¹ complexes, all prevent this DDT pathway. Our results demonstrate that the p53-mediated DDT requires the formation of a DNA binding-proficient p53 tetramer, recruitment of such tetramer to RPA-coated forks and p53 complex formation with POLĪ¹. Importantly, our mutational analysis demonstrates that transcriptional transactivation is dispensable for the POLĪ¹-mediated DDT pathway, which we show protects against DNA replication damage from endogenous and exogenous sources.Fil: Biber, Stephanie. Universitat Ulm; AlemaniaFil: Pospiech, Helmut. Fritz Lipmann Institute; Alemania. University Of Oulu (oy);Fil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones CientĆficas y TĆ©cnicas. Oficina de CoordinaciĆ³n Administrativa Parque Centenario. Instituto de Investigaciones BioquĆmicas de Buenos Aires. FundaciĆ³n Instituto Leloir. Instituto de Investigaciones BioquĆmicas de Buenos Aires; ArgentinaFil: WiesmĆ¼ller, Lisa. Universitat Ulm; Alemani
The solution structure of the amino-terminal domain of human DNA polymerase Īµ subunit B is homologous to C-domains of AAA+ proteins
DNA polymerases Ī±, Ī“ and Īµ are large multisubunit complexes that replicate the bulk of the DNA in the eukaryotic cell. In addition to the homologous catalytic subunits, these enzymes possess structurally related B subunits, characterized by a carboxyterminal calcineurin-like and an aminoproximal oligonucleotide/oligosaccharide binding-fold domain. The B subunits also share homology with the exonuclease subunit of archaeal DNA polymerases D. Here, we describe a novel domain specific to the N-terminus of the B subunit of eukaryotic DNA polymerases Īµ. The N-terminal domain of human DNA polymerases Īµ (Dpoe2NT) expressed in Escherichia coli was characterized. Circular dichroism studies demonstrated that Dpoe2NT forms a stable, predominantly Ī±-helical structure. The solution structure of Dpoe2NT revealed a domain that consists of a left-handed superhelical bundle. Four helices are arranged in two hairpins and the connecting loops contain short Ī²-strand segments that form a short parallel sheet. DALI searches demonstrated a striking structural similarity of the Dpoe2NT with the Ī±-helical subdomains of ATPase associated with various cellular activity (AAA+) proteins (the C-domain). Like C-domains, Dpoe2NT is rich in charged amino acids. The biased distribution of the charged residues is reflected by a polarization and a considerable dipole moment across the Dpoe2NT. Dpoe2NT represents the first C-domain fold not associated with an AAA+ protein
BRCA1 mislocalization leads to aberrant DNA damage response in heterozygous ABRAXAS1 mutation carrier cells
Peer reviewe
The randomized shortened dental arch study (RaSDA): design and protocol
<p>Abstract</p> <p>Background</p> <p>Various treatment options for the prosthetic treatment of jaws where all molars are lost are under discussion. Besides the placement of implants, two main treatment types can be distinguished: replacement of the missing molars with removable dental prostheses and non-replacement of the molars, i.e. preservation of the shortened dental arch. Evidence is lacking regarding the long-term outcome and the clinical performance of these approaches. High treatment costs and the long time required for the treatment impede respective clinical trials.</p> <p>Methods/design</p> <p>This 14-center randomized controlled investigator-initiated trial is ongoing. Last patient out will be in 2010. Patients over 35 years of age with all molars missing in one jaw and with at least both canines and one premolar left on each side were eligible. One group received a treatment with removable dental prostheses for molar replacement (treatment A). The other group received a treatment limited to the replacement of all missing anterior and premolar teeth using fixed bridges (treatment B). A pilot trial with 32 patients was carried out. Two hundred and fifteen patients were enrolled in the main trial where 109 patients were randomized for treatment A and 106 for treatment B. The primary outcome measure is further tooth loss during the 5-year follow-up. The secondary outcome measures encompassed clinical, technical and subjective variables. The study is funded by the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG WA 831/2-1, 2-2, 2-3, 2-4, 2-5).</p> <p>Discussion</p> <p>The particular value of this trial is the adaptation of common design components to the very specific features of complex dental prosthetic treatments. The pilot trial proved to be indispensable because it led to a number of adjustments in the study protocol that considerably improved the practicability. The expected results are of high clinical relevance and will show the efficacy of two common treatment approaches in terms of oral health. An array of secondary outcome measures will deliver valuable supplementary information. If the results can be implemented in the clinical practice, the daily dental care should strongly profit thereof.</p> <p>Trial registration</p> <p>The trial is registered at ClinicalTrials.gov under ISRCTN68590603 (pilot trial) and ISRCTN97265367 (main trial).</p
The role of DNA polymerases, in particular DNA polymerase Īµ in DNA repair and replication
Abstract
Analysis of the primary structure of DNA polymerase Īµ B subunit
defined similarities to B subunits of eukaryotic DNA polymerases Ī±,
Ī“ and Īµ as well as the small subunits of DNA polymerase DI of
Euryarchaeota. Multiple sequence alignment of these proteins revealed the
presence of 12 conserved motifs and defined a novel protein superfamily.
The members of the B subunit family share a common domain architecture,
suggesting a similar fold, and arguing for a conserved function among
these proteins.
The contribution of human DNA polymerase Īµ to nuclear DNA
replication was studied using the antibody K18 that specifically inhibits
the activity of this enzyme in vitro. This antibody
significantly inhibited DNA synthesis both when microinjected into nuclei
of exponentially growing human fibroblasts and in isolated HeLa cell
nuclei, but did not inhibit SV40 DNA replication in
vitro. These results suggest that the human DNA polymerase
Īµ contributes substantially to the replicative synthesis of DNA and
emphasises the differences between cellular replication and viral model
systems.
The human DNA polymerases Īµ and Ī“ were found capable of
gap-filling DNA synthesis during nucleotide excision repair in
vitro. Both enzymes required PCNA and the clamp loader RFC, and
in addition, polymerase Ī“ required Fen-1 to prevent excessive
displacement synthesis. Nucleotide excision repair of a defined DNA lesion
was completely reconstituted utilising largely recombinant proteins, only
ligase I and DNA polymerases Ī“ and Īµ provided as highly purified
human enzymes. This system was also utilised to study the role of the
transcription factor II H during repair.
Human non-homologous end joining of model substrates with different
DNA end configurations was studied in HeLa cell extracts. This process
depended partially on DNA synthesis as an aphidicolin-dependent DNA
polymerase was required for the formation of a subset of end joining
products. Experiments with neutralising antibodies reveal that DNA
polymerase Ī± but not DNA polymerases Ī² or Īµ, may represent
this DNA polymerase activity. Our results indicate that DNA synthesis
contributes to the stability of DNA ends, and influences both the
efficiency and outcome of the end joining event. Furthermore, our results
suggest a minor role of PCNA in non-homologous end joining
DNA Polymerase e - More Than a Polymerase
This paper presents a comprehensive review of the structure and function of DNA polymerase e. Together with DNA polymerases a and d, this enzyme replicates the nuclear DNA in the eukaryotic cell. During this process, DNA polymerase a lays down RNA-DNA primers that are utilized by DNA polymerases d and e for the bulk DNA synthesis. Attempts have been made to assign these two enzymes specifically to the synthesis of the leading and the lagging strand. Alternatively, the two DNA polymerases may be needed to replicate distinct regions depending on chromatin structure. Surprisingly, the essential function of DNA polymerase e does not depend on its catalytic activity, but resides in the nonenzymatic carboxy-terminal domain. This domain not only mediates the interaction of the catalytic subunit with the three smaller regulatory subunits, but also links the replication machinery to the S phase checkpoint. In addition to its role in DNA replication, DNA polymerase e fulfils roles in the DNA synthesis step of nucleotide excision and base excision repair, and has been implicated in recombinational processes in the cell
DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding
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
Structural organization and splice variants of the POLE1 gene encoding the catalytic subunit of human DNA polymerase Īµ
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