Purified human BRCA2 stimulates RAD51-mediated recombination.

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells

Estudio del desensamblaje in vitro y análisis estructura-función de cavidades de la cápsida del virus diminuto del ratón

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura : 20-10-2005Sphencal virus capsids are large, multimeric proteins and constitute attractive models for the study of the assoeiation, stabiity and disassembly of very large protein complexes. In addition, they provide good oppominities to understand finely tuned shucturehinction relationships in viral particles, and to develop more stable vaccines and new antiviral approaches based on the inhibition of assembly or uncoating. In this doctoral thesis we have analyzed in viíro the disassembly of the MVM capsid, and carried out a shucture-function analysis of the role of cavities and a bwied charge located within the hydrophobic core of each capsid subunit on capsid assembly and stabiiity and viral function. The specific results obtained can be summarized as fouows. 1. Biophysical and biochemical Vi viíro analyses of the disassembly of the MVM capsid reveal a complex pathway that may involve several steps: a reversible conformational rearrangement associated, but not limited to the externalization of the N-terminal segment of a fraction of the capsid subunits; the irreversible dissociation of the capsid into an intermediate; and the dissociatioddenaturation of the latter to yield a molten globulelike monomeric form. 2. Determination by intrinsic Trp fluorescence of the transition temperature of the specific capsid dissociation step has been validated as a reliable and acauate procedure to analyze the effect of mutations or conditions on the relative stability of the MVM capsid against thermal dissociation. 3. Insertion of heterologous epitopes in four solvent-exposed loops at the MVM capsid surface led to senous defects in capsid assembly en4 in most cases, to a reduction in capsid stability, even though the insertions were made in pooitions that are, presumably, the most tolerant from a structural point of view. Two out of nine conservative point replacements of residues located within surface loops, and involved in very few or no interactions, led also to defe& in capsid assembly. 4. The region surrounding an ensemble of conspicuous cavities within the hydrophobic core of each MVM capsid subunit is able to accept the introduction of as many as three extra methylenes or equivalent-sized groups, or the removal of as many as fow groups, without any signiscant effect on capsid assembly or stability against thermal dissociation. Assembly was impaired only when fow or more groups were introduced, or six or more groups removed 5. Such shucairal tolerance to mutation around cavities within the protein core may no1 imply a similar functional tolerance. A combinatorial mutagenesis approach suggests that MVM requires, for optimum biological fítness, the presence of very specific residues that preserve the size and shape of the largest cavities within the core of each subunit This obsmration may explain the high degree of conservation of those residues in MVM and evolutionarily related pawoviruses. 6. Assembly of the MVM capsid requires aiso the presence of the negative charge of a carboxylate buried within the largest caviiy in the protein core, and speciñcaily located at position 115. Viral DNA encapsidation specifically requires, in addition, the presence of aspartate, and not glutamate, at position 115. This justifies the absolute conservation of AspllS in MVM and other parvoviruses

Preface-Homologous Recombination

DNA double-strand breaks (DSBs) are the most harmful lesions to DNA in the cell. To cope with these insults, all organisms have devised two main types of evolutionary conserved mechanisms for their repair, homologous recombination (HR), and non-homologous end joining (NHEJ). The first one operates predominantly during the S/G2 phase of the cell cycle, when the sister chromatid is available for repair. Because HR requires homology for repair, this pathway is considered essentially error-free. NHEJ is the pathway of choice in the other phases of the cell cycle, including G1. This pathway is generally faithful but can be prone to errors. Most spontaneous DNA breaks arising in somatic cells occur randomly as a consequence of DNA replication failure caused by either DNA lesions or generated by obstacles that impede the progression of the replication fork (e.g., protein-bound to DNA, DNA secondary structures, replication–transcription conflicts, etc.). For this reason, HR is a major DNA repair pathway during S/G2 phases of the cell cycle. Thus, HR is intimately ligated to the prevention of genome instability in replicating somatic cells. In meiotic cells however, DSBs are developmentally controlled by the action of specific endonucleases where HR is essential; gametogenesis is not possible in the absence of HR. Genome instability and in particular defective HR is a common feature of a number of genetic diseases including cancer. Defects in HR in meiotic cells can lead to birth defects such as Down syndrome. Considering the relevance of HR as one of the major DSB repair pathways in mitotically cycling cells, as well as its essential role in meiosis, understanding the molecular mechanisms and factors that participate in HR is of key importance in Molecular Biology and Biomedicine. In this book, we compile a series of laboratory protocols covering the analysis of different steps of the homologous recombination process from the genetic, molecular biology, and cell biology perspectives. As these steps are very well conserved through evolution, taking advantage of different model organisms have led to accelerated discoveries in this field. Thus, when appropriate, some of the protocols we present here are explained in the context of more than one model system. We hope this book will facilitate the use of both classical and more recent approaches to answer specific questions on HR mechanisms as well as to decipher the function of novel factors involved in HR. We expect that this compilation of protocols elaborated by leading experts in the field will be useful not only to the scientific community working in genome integrity but also to scientists working in other areas such as cancer biology or cell cycle with renovated interests in HR and DSB repair

Purified human BRCA2 stimulates RAD51-mediated recombination.

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer

Missense Variants of Uncertain Significance: A Powerful Genetic Tool for Function Discovery with Clinical Implications

The breast cancer susceptibility gene BRCA2 encodes a multifunctional protein required for the accurate repair of DNA double-strand breaks and replicative DNA lesions. In addition, BRCA2 exhibits emerging important roles in mitosis. As a result, mutations in BRCA2 may affect chromosomal integrity in multiple ways. However, many of the BRCA2 mutations found in breast cancer patients and their families are single amino acid substitutions, sometimes unique, and their relevance in cancer risk remains difficult to assess. In this review, we focus on three recent reports that investigated variants of uncertain significance (VUS) located in the N-terminal region of BRCA2. In this framework, we make the case for how the functional evaluation of VUS can be a powerful genetic tool not only for revealing novel aspects of BRCA2 function but also for re-evaluating cancer risk. We argue that other functions beyond homologous recombination deficiency or “BRCAness” may influence cancer risk. We hope our discussion will help the reader appreciate the potential of these functional studies in the prevention and diagnostics of inherited breast and ovarian cancer. Moreover, these novel aspects in BRCA2 function might help find new therapeutic strategies

A new interaction between BRCA2 and DDX5 promotes the repair of DNA breaks at transcribed chromatin

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2T207A missense variant found in breast cancer patients

1H, 13C and 15N backbone resonance assignment of the human BRCA2 N-terminal region

International audienceThe Breast Cancer susceptibility protein 2 (BRCA2) is involved in mechanisms that maintain genome stability, including DNA repair, replication and cell division. These functions are ensured by the folded C-terminal DNA binding domain of BRCA2 but also by its large regions predicted to be disordered. Several studies have shown that disordered regions of BRCA2 are subjected to phosphorylation, thus regulating BRCA2 interactions through the cell cycle. The N-terminal region of BRCA2 contains two highly conserved clusters of phosphorylation sites between amino acids 75 and 210. Upon phosphorylation by CDK, the cluster 1 is known to become a docking site for the kinase PLK1. The cluster 2 is phosphorylated by PLK1 at least at two positions. Both of these phosphorylation clusters are important for mitosis progression, in particular for chromosome segregation and cytokinesis. In order to identify the phosphorylated residues and to characterize the phosphorylation sites preferences and their functional consequences within BRCA2 N-terminus, we have produced and analyzed the BRCA2 fragment from amino acid 48 to amino acid 284 (BRCA248-284). Here, we report the assignment of 1H, 15N, 13CO, 13Cα and 13Cβ NMR chemical shifts of this region. Analysis of these chemical shifts confirmed that BRCA248-284 shows no stable fold: it is intrinsically disordered, with only short, transient α-helices