Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.

DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse.

Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases.

In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.

Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity.

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.

Sex and the single cell: meiosis in yeast.

Recent studies of Saccharomyces cerevisiae have significantly advanced our understanding of the molecular mechanisms of meiotic chromosome behavior. Structural components of the synaptonemal complex have been identified and studies of mutants defective in synapsis have provided insight into the role of the synaptonemal complex in homolog pairing, genetic recombination, crossover interference, and meiotic chromosome segregation. There is compelling evidence that most or all meiotic recombination events initiate with double-strand breaks. Several intermediates in the double-strand break repair pathway have been characterized and mutants blocked at different steps in the pathway have been identified. With the application of genetic, molecular, cytological, and biochemical methods in a single organism, we can expect an increasingly comprehensive and unified view of the meiotic process.

Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8.

The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large (approximately 460 kDa) catalytic polypeptide (DNA-PKcs) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PKcs to DNA. A 41-kbp segment of the DNA-PKcs gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PKcs gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells.

Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Expression of ATM, p53, and the MRE11-Rad50-NBS1 complex in myoepithelial cells from benign and malignant proliferations of the breast

Aims: To analyse the expression of proteins involved in DNA double strand break detection and repair in the luminal and myoepithelial compartments of benign breast lesions and malignant breast tumours with myoepithelial differentiation. Methods: Expression of the ataxia telangiectasia (ATM) and p53 proteins was immunohistochemically evaluated in 18 benign and malignant myoepithelial tumours of the breast. Fifteen benign breast lesions with prominent myoepithelial compartment were also evaluated for these proteins, in addition to those in the MRE11-Rad50-NBS1 (MRN) complex, and the expression profiles were compared with those seen in eight independent non-cancer (normal breast) samples and in the surrounding normal tissues of the benign and malignant tumours examined. Results: ATM expression was higher in the myoepithelial compartment of three of 15 benign breast lesions and lower in the luminal compartment of eight of these lesions compared with that found in the corresponding normal tissue compartments. Malignant myoepithelial tumours overexpressed ATM in one of 18 cases. p53 was consistently negative in benign lesions and was overexpressed in eight of 18 malignant tumours. In benign breast lesions, expression of the MRN complex was significantly more reduced in myoepithelial cells (up to 73%) than in luminal cells (up to 40%) (p = 0.0005). Conclusions: Malignant myoepithelial tumours rarely overexpress ATM but are frequently positive for p53. In benign breast lesions, expression of the MRN complex was more frequently reduced in the myoepithelial than in the luminal epithelial compartment, suggesting different DNA repair capabilities in these two cell types.

Novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtDNA recombination in the chigger mite, Leptotrombidium pallidum

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

Topoisomerase 1 (Top1)-associated Genome Instability in Yeast: Effects of Persistent Cleavage Complexes or Increased Top1 Levels

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.

Genetic Analysis of Mitotic Recombination in Saccharomyces cerevisiae

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.

Biochemical Characterization of Human Exonuclease 1 Protein-protein Interactions

Human Exonuclease 1 (Exo1) plays important roles in numerous DNA metabolic/repair pathways including DNA mismatch repair, DNA double strand break repair, Okazaki fragment maturation etc. The nuclease activity of Exo1 is tightly regulated in vivo. The regulation of Exo1 in different pathways is achieved by interactions with different protein partners. The focus of this dissertation will be on characterization of Exo1 interactions with traditional protein partners and providing experimental evidences for new Exo1 interactions.

Molecular cloning, biochemical assays, collaborative nuclear magnetic resonance and X-ray crystallography have been employed to study Exo1 interactions with protein partners. This work contains: (i) the experimental evidence for new Exo1 interactions, and (ii) the detailed characterization of Exo1 interactions with PCNA, MLH1 and MutSα/β.

Taken together, the research progress presented in this dissertation further advances our understanding of traditional Exo1 interaction network and probably provides new insights to new functions and new regulations of Exo1.

Análise da expressão de APE1 após estresse oxidativo induzido em células proficientes e deficientes na via de reparo por excisão de nucleotídeos.

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

INDUCTION OF DNA STRAND BREAKS AND CROSS-LINKS BY THE NEW ANTICANCER AGENT 3,6-DIAZIRIDINYL-2,5-BIS(CARBOETHOXYAMINO) -1,4-BENZOQUINONE

The DNA breakage effect of the anticancer agent 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone (AZQ, NSC-182986) on bacteriophage PM2 DNA was investigated using agarose gel electrophoresis. AZQ caused both single-stranded and double-stranded breaks after reduction with NaBH(,4), but it was not active in the native state. At 120 (mu)M, it degraded 50% of the closed circular form I DNA into 40% form II DNA (single-stranded break) and 10% form III DNA (double-stranded break). It produced a dose-response breakage between 1 (mu)M and 320 (mu)M. The DNA breakage exhibited a marked pH dependency. At 320 (mu)M, AZQ degraded 80% and 60% of form I DNA at pH 4 and 10 respectively, but none between pH 6 to 8. The DNA breakage at physiologic pH was greatly enhanced when 10 (mu)M cupric sulfate was included in the incubation mixture. The DNA strand scission was inhibited by catalase, glutathione, KI, histidine, Tiron, and DABCO. These results suggest that the DNA breakage may be caused by active oxygen metabolites including hydroxyl free radical. The bifunctional cross-linking activity of reduced AZQ on isolated calf thymus DNA was investigated by ethidium fluorescence assay. The cross-linking activity exhibited a similar pH dependency; highest in acidic and alkaline pH, inactive under neutral conditions. Using the alkaline elution method, we found that AZQ induced DNA single-stranded breaks in Chinese hamster ovary cells treated with 50 (mu)M of AZQ for 2 hr. The single-stranded break frequencies in rad equivalents were 17 with 50 (mu)M and 140 with 100 (mu)M of AZQ. In comparison, DNA cross-links appeared in cells treated with only 1 to 25 (mu)M of AZQ for 2 hr. The cross-linking frequencies in rad equivalents were 39 and 90 for 1 and 5 (mu)M of AZQ, respectively. Both DNA-DNA and DNa-protein cross-links were induced by AZQ in CHO cells as revealed by the proteinas K digestion assay. DNA cross-links increased within the first 4 hr of incubation in drug-free medium and slightly decreased by 12 hr, and most of the cross-links disappeared after cells were allowed to recovered for 24 hr.^ By electrochemical analysis, we found that AZQ was more readily reduced at acidic pH. However, incubation of AZQ with NaBH(,4) at pH 7.8 or 10, but not at 4, produced superoxide anion. The opening of the aziridinyl rings of AZQ at pH 4 was faster in the presence of NaBH(,4) than in its absence; no ring-opening was detected at pH 7.8 regardless of the inclusion of NaBH(,4). . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

On The Consistency Of Monte Carlo Track Structure Dna Damage Simulations.

Monte Carlo track structures (MCTS) simulations have been recognized as useful tools for radiobiological modeling. However, the authors noticed several issues regarding the consistency of reported data. Therefore, in this work, they analyze the impact of various user defined parameters on simulated direct DNA damage yields. In addition, they draw attention to discrepancies in published literature in DNA strand break (SB) yields and selected methodologies. The MCTS code Geant4-DNA was used to compare radial dose profiles in a nanometer-scale region of interest (ROI) for photon sources of varying sizes and energies. Then, electron tracks of 0.28 keV-220 keV were superimposed on a geometric DNA model composed of 2.7 × 10(6) nucleosomes, and SBs were simulated according to four definitions based on energy deposits or energy transfers in DNA strand targets compared to a threshold energy ETH. The SB frequencies and complexities in nucleosomes as a function of incident electron energies were obtained. SBs were classified into higher order clusters such as single and double strand breaks (SSBs and DSBs) based on inter-SB distances and on the number of affected strands. Comparisons of different nonuniform dose distributions lacking charged particle equilibrium may lead to erroneous conclusions regarding the effect of energy on relative biological effectiveness. The energy transfer-based SB definitions give similar SB yields as the one based on energy deposit when ETH ≈ 10.79 eV, but deviate significantly for higher ETH values. Between 30 and 40 nucleosomes/Gy show at least one SB in the ROI. The number of nucleosomes that present a complex damage pattern of more than 2 SBs and the degree of complexity of the damage in these nucleosomes diminish as the incident electron energy increases. DNA damage classification into SSB and DSB is highly dependent on the definitions of these higher order structures and their implementations. The authors' show that, for the four studied models, different yields are expected by up to 54% for SSBs and by up to 32% for DSBs, as a function of the incident electrons energy and of the models being compared. MCTS simulations allow to compare direct DNA damage types and complexities induced by ionizing radiation. However, simulation results depend to a large degree on user-defined parameters, definitions, and algorithms such as: DNA model, dose distribution, SB definition, and the DNA damage clustering algorithm. These interdependencies should be well controlled during the simulations and explicitly reported when comparing results to experiments or calculations.