EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

<p>Abstract</p> <p>Background</p> <p>Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand.</p> <p>Results</p> <p>EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data.</p> <p>Conclusion</p> <p>EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from <url>http://www.ezarray.com/</url>.</p

Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution

Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity

RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination

Clinical Impact Of TP53 Gene Mutations In Diffuse Large B-cell Lymphoma (DLBCL): An International DLBCL Rituxan-CHOP Consortium Program Study

Oral SessionMutations of the TP53 tumor suppressor gene are associated with a poor clinical outcome in DLBCL patients treated with CHOP. The impact of TP53 mutations on clinical outcome of DLBCL patients treated with Rituxan-CHOP has not been comprehensively analyzed. The purpose of this study was to analyze the frequency and type of TP53 mutations in Rituxan-CHOP treated DLBCL patients from twenty-two medical centers, and to correlate these with clinical outcome. TP53 mutations were identified in 138/604 (22.7%) Rituxan-CHOP treated DLBCL cases and included missense (n=133), nonsense (n=16), splice site (n=9) and frameshift (n=1) mutations. The presence of any TP53 mutation correlated with poor overall survival (OS) with a median OS of 50 months in the TP53 mutation group versus 69 months in the wild-type group (wt-TP53, P=0.0042). Seventy-three of 138 cases (53%) had mutations in the DNA binding domains of the TP53 gene, which were found to be the most important predictor of poor OS (P=0.0044). In contrast, mutations in the non-DNA binding domains did not correlate with poor OS (P=0.157). Overexpression of p53 protein significantly correlated with only TP53 missense mutations (P=0.002), but not with other types of TP53 mutations, while TP53 deletion did not correlate with mutation or OS. In comparison to our previous series of patients treated only with CHOP, Rituxan-CHOP regimen improved OS in both wt-TP53 and TP53 mutated groups. The 5-year survival rate was 42% in patients with any TP53 mutation (median survival=50 months) and 41% in patients with the DNA-binding domain mutations (median survival=49 months) compared to 52% for those with wt-TP53 (median survival=69 months). The complete remission rate was 51% in patients with any TP53 mutation and 44% in patients with the DNA-binding domain mutations, compared to 77% for those with wt-TP53. However, the clinical outcome and treatment response to the Rituxan-CHOP varied in patients with mutations in different regions of the DNA-binding domains. Patients with mutations in the DNA minor binding groove motif (Loop L3, 17% of all mutations) had significantly decreased median OS (17 months) when compared to patients with Loop L2 (16% of all mutations) or loop-sheet-helix motifs (Loop L1-S10-H2, 20% of all mutations) with median OS of 49 and 50 months, respectively. In contrast to our previous CHOP series study, median survival was significantly improved for Rituxan-CHOP treated DLBCL patients with mutations in the loop-sheet-helix motifs (43 months). Multivariate analysis confirmed that TP53 mutations and activated B-cell-like (ABC)/germinal center B-cell-like (GCB) subtype classification were independent predictors of OS with a hazard ratio of 0.69 (GCB vs ABC, 95% CI 0.49-0.98) and 1.60 (TP53 vs wt-TP53, 95% CI 1.10-2.31), respectively. Similar to our previous CHOP study, the TP53 mutation profile, regardless of location, was found to stratify GCB-DLBCL, but not ABC-DLBCL, into molecularly distinct subsets with different clinical outcomes in Rituxan-CHOP treated DLBCL patients. This study demonstrates the importance of TP53 mutational profile for predicting clinical outcome. Elucidation of the roles of specific TP53 domain mutations, as documented in our study, will help in refining prognostic models for DLBCL patients treated with either the CHOP or Rituxan-CHOP regimen. These findings also provide the rationale and strategies for p53 targeted therapeutic intervention in DLBCL patients. © 2009 by The American Society of Hematolog

RecA bundles mediate homology pairing between distant sisters during DNA break repair

DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions