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Biomedical subjects

Claire Wyman

Publications and source records attributed to Claire Wyman.

At least 19 recordsLinked to original sources
PubMed2006

The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks.

Repair of interstrand crosslinks (ICLs) requires multiple-strand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible for producing these intermediates are unknown. Here we show that Mus81, a component of the Mus81-Eme1 structure-specific endonuclease, is involved in generating the ICL-induced DSBs in mouse embryonic stem (ES) cells in S phase. Given the DNA junction cleavage specificity of Mus81-Eme1 in vitro, DNA damage-stalled replication forks are suitable in vivo substrates. Interestingly, generation of DSBs from replication forks stalled due to DNA damage that affects only one of the two DNA strands did not require Mus81. Furthermore, in addition to a physical interaction between Mus81 and the homologous recombination protein Rad54, we show that Mus81(-/-) Rad54(-/-) ES cells were as hypersensitive to ICL agents as Mus81(-/-) cells. We propose that Mus81-Eme1- and Rad54-mediated homologous recombination are involved in the same DNA replication-dependent ICL repair pathway.

Animals↗
PubMed2006

Architecture of a polycomb nucleoprotein complex.

Polycomb group (PcG) epigenetic silencing proteins act through cis-acting DNA sequences, named Polycomb response elements (PREs). Within PREs, Pleiohomeotic (PHO) binding sites and juxtaposed Pc binding elements (PBEs) function as an integrated DNA platform for the synergistic binding of PHO and the multisubunit Polycomb core complex (PCC). Here, we analyzed the architecture of the PHO/PCC/PRE nucleoprotein complex. DNase I footprinting revealed extensive contacts between PHO/PCC and the PRE. Scanning force microscopy (SFM) in combination with DNA topological assays suggested that PHO/PCC wraps the PRE DNA around its surface in a constrained negative supercoil. These features are difficult to reconcile with the simultaneous presence of nucleosomes at the PRE. Indeed, chromatin immunoprecipitations (ChIPs) and nuclease mapping demonstrated that PREs are nucleosome depleted in vivo. We discuss the implications of these findings for models explaining PRE function.

Animals↗
PubMed2006

Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers.

Nucleoprotein filament formation by recombinases is central to homologous recombination. To follow this process, we used fluorescent human Rad51 recombinase to visualize the interactions with double-stranded DNA (dsDNA). Fluorescence imaging revealed that Rad51 filament formation on dsDNA initiates from multiple nucleation points, resulting in Rad51-dsDNA nucleoprotein filaments interspersed with regions of bare DNA. The elastic properties of such heterogeneously coated DNA molecules were assessed by combining force-extension measurements using optical traps with fluorescence microscopy. This combination of single-molecule techniques allows discrimination of segments within an individual DNA molecule and determination of their elastic properties. The nonfluorescent zones of DNA-Rad51 constructs showed the well-known (over)stretching behavior of bare DNA. In contrast, the fluorescent, Rad51-coated zones did not overstretch and Rad51 remained stably bound in a structure that was approximately 50% longer than bare DNA. These results illustrate the power of adding sensitive fluorescence imaging to optical tweezers instrumentation.

DNA↗
PubMed2006

Multiple interlinked mechanisms to circumvent DNA replication roadblocks.

DNA replication is a fragile process, since unavoidable lesions in the template DNA cause replicative polymerases to stall, posing a serious threat to genome integrity. Homologous recombination, translesion DNA synthesis and de novo reinitiation of DNA synthesis ensure robust replication by navigating it passed damaged DNA. In this review, we highlight the relationship between these three processes.

Animals↗
PubMed2006

DNA double-strand break repair: all's well that ends well.

Breaks in both DNA strands are a particularly dangerous threat to genome stability. At a DNA double-strand break (DSB), potentially lost sequence information cannot be recovered from the same DNA molecule. However, simple repair by joining two broken ends, though inherently error prone, is preferable to leaving ends broken and capable of causing genome rearrangements. To avoid DSB-induced genetic disinformation and disruption of vital processes, such as replication and transcription, cells possess robust mechanisms to repair DSBs. Because all breaks are not created equal, the particular repair mechanism used depends largely on what is possible and needed based on the structure of the broken DNA. We argue that although categorizing different DSB repair mechanisms along pathways and subpathways can be conceptually useful, in cells flexible and reversible interactions among DSB repair factors form a web from which a nonpredetermined path to repair for any number of different DNA breaks will emerge.

Animals↗
PubMed2005

Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA.

The human Rad50/Mre11/Nbs1 complex (hR/M/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function results from its ability to tether broken DNA molecules. hR/M/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their apices can self-associate. The flexibility of the coiled coils allows their apices to adopt an orientation favourable for interaction. However, this also allows interaction between the tips of two coiled coils within the same complex, which competes with and frustrates the intercomplex interaction required for DNA tethering. Here we show that the dynamic architecture of hR/M/N is markedly affected by DNA binding. DNA binding by the hR/M/N globular domain leads to parallel orientation of the coiled coils; this prevents intracomplex interactions and favours intercomplex associations needed for DNA tethering. The hR/M/N complex thus is an example of a biological nanomachine in which binding to its ligand, in this case DNA, affects the functional conformation of a domain located 50 nm distant.

Acid Anhydride Hydrolases↗
PubMed2005

Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore.

The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex.

Cell Cycle Proteins↗
PubMed2005

Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function.

Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.

Adenosine Triphosphate↗
PubMed2005

Analysis of scanning force microscopy images of protein-induced DNA bending using simulations.

Bending of DNA is a feature essential to the function of many DNA-binding proteins. Bending angles can be estimated with a variety of techniques, but most directly from images obtained using scanning force microscopy (SFM). Direct measurement of the bending angle using a tangent method often produces angles that deviate significantly from values obtained using other techniques. Here, we describe the application of SFM in combination with simulations of DNA as a means to estimate protein-induced bending angles in a reliable and unbiased fashion. In this manner, we were able to obtain accurate estimates for the bending angles induced by nuclear factor I, octamer-binding transcription factor 1, the human XPC-Rad23B complex and integration host factor [correction]

CCAAT-Enhancer-Binding Proteins↗
PubMed2005

Torque-limited RecA polymerization on dsDNA.

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA-DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 +/- 0.7 k(b)T. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.

Biopolymers↗
PubMed2005

DNA polymerase V and RecA protein, a minimal mutasome.

A hallmark of the Escherichia coli SOS response is the large increase in mutations caused by translesion synthesis (TLS). TLS requires DNA polymerase V (UmuD'2C) and RecA. Here, we show that pol V and RecA interact by two distinct mechanisms. First, pol V binds to RecA in the absence of DNA and ATP and second, through its UmuD' subunit, requiring DNA and ATP without ATP hydrolysis. TLS occurs in the absence of a RecA nucleoprotein filament but is inhibited in its presence. Therefore, a RecA nucleoprotein filament is unlikely to be required for SOS mutagenesis. Pol V activity is severely diminished in the absence of RecA or in the presence of RecA1730, a mutant defective for pol V mutagenesis in vivo. Pol V activity is strongly enhanced with RecA mutants constitutive for mutagenesis in vivo, suggesting that RecA is an obligate accessory factor that activates pol V for SOS mutagenesis.

Adenosine Triphosphate↗
PubMed2004

Dynamics of relative chromosome position during the cell cycle.

The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photobleaching of GFP-labeled histone H2B, local UV-marked DNA, and photobleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers, suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods.

Animals↗
PubMed2004

NFI and Oct-1 bend the Ad5 origin in the same direction leading to optimal DNA replication.

Two cellular transcription factors, nuclear factor I (NFI) and octamer binding protein (Oct-1), bind simultaneously to their recognition sequences in the Ad5 origin of replication thereby enhancing initiation. Using scanning force microscopy we have previously shown that NFI induces a 60 degrees bend in the origin DNA. Here we demonstrate that Oct-1 induces a 42 degrees bend in the origin DNA. Simultaneous binding of NFI and Oct-1 induces an 82 degrees collective bend suggesting that both bends are oriented towards each other. In functional replication assays we further demonstrate that this extensive DNA bending leads to a synergistic enhancement of DNA replication. We propose that collective DNA bending induced by NFI and Oct-1 facilitates the optimal assembly of the preinitiation complex and plays an important role in the stimulatory mechanism of NFI and Oct-1 in replication.

Adenoviridae↗
PubMed2004

The CSB protein actively wraps DNA.

The CSB protein is a member of the SWI2/SNF2 family of ATP-dependent chromatin remodeling factors and is essential for transcription-coupled DNA repair. The role of CSB in this DNA repair process is unclear, but the protein was found to remodel nucleosomes and alter DNA double helix conformation upon binding. Elucidating the nature of the change in DNA structure induced by CSB is of great interest for understanding the CSB mechanism of action. We analyzed the CSB.DNA complex by scanning force microscopy and measured a shortening of DNA contour length upon CSB binding in the presence of ATP. This DNA length reduction most likely results from DNA wrapping around the protein. Shorter DNA molecules were observed more frequently in the presence of non-hydrolyzable ATP analogues. These results suggest that DNA wrapping depends on ATP binding, whereas ATP hydrolysis results in unwrapping. We also provide evidence suggesting that CSB binds DNA as a dimer. DNA wrapping and unwrapping allows CSB to actively alter the DNA double helix conformation, which could influence nucleosomes and other protein-DNA interactions.

Adenosine Triphosphate↗
PubMed2004

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization.

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

Amino Acid Motifs↗
PubMed2004

Homologous recombination: down to the wire.

Exchange of strands between homologous DNA molecules is catalyzed by evolutionarily conserved recombinases. These proteins can occur in different quaternary arrangements: rings or helical filaments. Recent results reveal that recombinase function follows from the filamentous form.

Adenosine Triphosphate↗