Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing
DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'–3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11–Rad50–Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.
Plasma DNA Is More Reliable than Carcinoembryonic Antigen for Diagnosis of Recurrent Esophageal Cancer
Background
Carcinoembryonic antigen (CEA) and plasma DNA are known to be elevated in patients with esophageal cancer and are higher in patients with disseminated disease. The sensitivity and specificity of these markers in the diagnosis of recurrent esophageal cancer have not been compared.
Study Design
Plasma DNA was measured using polymerase chain reaction in 45 patients with esophageal cancer and 44 asymptomatic volunteers. The 95th percentile (19 ng /mL) in the volunteers was used to define normal. Thirty-nine patients had localized cancer and underwent resection, and six had disseminated disease at operation. Plasma DNA was measured preoperatively in all patients, with serum CEA measured in 31. Plasma DNA was measured sequentially during followup in 21 patients, including 7 who developed recurrence. CEA was measured in 14 of 21 patients who had sequential plasma DNA measured and in 6 of 7 patients with recurrence. CEA levels greater than 5.0 ng/mL were used as cut-off.
Results
Plasma DNA was more sensitive than CEA for detecting unresectable esophageal cancer (100% versus 40%), but it had a lower specificity (22% versus 89%).The positive predictive value (19% versus 40%) and negative predictive value (100% versus 89%) were similar for plasma DNA and serum CEA, respectively.
Plasma DNA was also more sensitive than CEA in detecting recurrent esophageal cancer (100% versus 33%). The specificity and positive predictive values were 100% for both tests, but the negative predictive values were higher for plasma DNA. Plasma DNA rose before there was clinical evidence of recurrence in 67% compared with only 17% for CEA.
Conclusions
Elevated plasma DNA is an extremely reliable indicator of the presence of recurrent disease, and, in the majority of patients, it rises before clinical evidence of recurrence. In contrast, a normal CEA should be interpreted cautiously, because it does not exclude recurrent disease.
Carcinoembryonic antigen (CEA) and plasma DNA are known to be elevated in patients with esophageal cancer and are higher in patients with disseminated disease. The sensitivity and specificity of these markers in the diagnosis of recurrent esophageal cancer have not been compared.
Study Design
Plasma DNA was measured using polymerase chain reaction in 45 patients with esophageal cancer and 44 asymptomatic volunteers. The 95th percentile (19 ng /mL) in the volunteers was used to define normal. Thirty-nine patients had localized cancer and underwent resection, and six had disseminated disease at operation. Plasma DNA was measured preoperatively in all patients, with serum CEA measured in 31. Plasma DNA was measured sequentially during followup in 21 patients, including 7 who developed recurrence. CEA was measured in 14 of 21 patients who had sequential plasma DNA measured and in 6 of 7 patients with recurrence. CEA levels greater than 5.0 ng/mL were used as cut-off.
Results
Plasma DNA was more sensitive than CEA for detecting unresectable esophageal cancer (100% versus 40%), but it had a lower specificity (22% versus 89%).The positive predictive value (19% versus 40%) and negative predictive value (100% versus 89%) were similar for plasma DNA and serum CEA, respectively.
Plasma DNA was also more sensitive than CEA in detecting recurrent esophageal cancer (100% versus 33%). The specificity and positive predictive values were 100% for both tests, but the negative predictive values were higher for plasma DNA. Plasma DNA rose before there was clinical evidence of recurrence in 67% compared with only 17% for CEA.
Conclusions
Elevated plasma DNA is an extremely reliable indicator of the presence of recurrent disease, and, in the majority of patients, it rises before clinical evidence of recurrence. In contrast, a normal CEA should be interpreted cautiously, because it does not exclude recurrent disease.
PlateSelect™ RNAi
PlateSelect™ RNAi are customizable 96-well RNAi duplexes. This format is ideal for researchers who want small amounts of RNAi duplexes in a ready-to-transfect plate format.
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RNAi duplexes in customizable 96-well plates
Select well orientation in 96-well plates and choose from Invitrogen’s Stealth™ or BLOCK-iT™ RNAi duplexes. The RNAi duplexes are provided at 1 nmol/well, and they are synthesized on demand, based on the most-up-to-date bioinformatics to reduce the chances of off-target effects.
Select the sequence and orientation of your duplexes to match your transfection protocol
Minimize off-target effects with on-demand, synthesized duplexes based on the most up-to-date bioinformatics
Order 1 to 96 duplexes per plate in a convenient 1 nmol scale—no minimum order
Choose Stealth™ RNAi chemically modified duplexes or BLOCK-iT™ RNAi duplexes using an easy-to-use interface
#rightRTE blockquote { margin-left: 10px; margin-right: 10px }
#mainRTE blockquote { margin-left: 20px; margin-right: 20px }
RNAi duplexes in customizable 96-well plates
Select well orientation in 96-well plates and choose from Invitrogen’s Stealth™ or BLOCK-iT™ RNAi duplexes. The RNAi duplexes are provided at 1 nmol/well, and they are synthesized on demand, based on the most-up-to-date bioinformatics to reduce the chances of off-target effects.
Select the sequence and orientation of your duplexes to match your transfection protocol
Minimize off-target effects with on-demand, synthesized duplexes based on the most up-to-date bioinformatics
Order 1 to 96 duplexes per plate in a convenient 1 nmol scale—no minimum order
Choose Stealth™ RNAi chemically modified duplexes or BLOCK-iT™ RNAi duplexes using an easy-to-use interface
BLOCK-iT™ Pol II miR RNAi Vector Services
BLOCK-iT? Pol II miR RNAi Vector ServicesDNA based or vector mediated RNA Interference (RNAi) is often used for long-term expression, hard to transfect cell lines or for inducible RNAi. Vector technologies allow you to:
Achieve transient or stable target knockdownPerform RNAi in any cell type – even hard to transfect, primary and non-diving cellsRegulate gene inhibition with inducible RNAi expressionStudy long-term gene knockdown
BLOCK-iT? Pol II miR RNAi vectors combine the benefits of traditional RNAi vectors – stable expression and the ability to use viral delivery – with capabilities for tissue-specific expression and multiple target knockdown from the same transcript. These vectors are designed to express artificial miRNAs that are engineered to have 100% homology to your target gene and result in target cleavage.
Table 1: BLOCK-iT? Pol II miR RNAi Entry VectorsmiR RNAi Entry Vector Advantages pcDNA?6.2- GW/miR Pol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors pcDNA?6.2- GW/EmGFP- miR Co-cistronic EmGFP reporter for easy tracking of miR RNAi expressionPol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors
Once your miR RNAi entry vector is generated, our RNAi services team will transfect it into mammalian cells to perform knockdown studies, chain multiple miR RNAi sequences together to knock down more than one target with the same vector, or transfer the miR RNAi sequence to another destination vector. Regardless of your vector choice, once expressed in a cell, the miR RNAi sequence induces an RNAi response resulting in knockdown of the targeted message.
Subcloning BLOCK-iT? Pol II miR RNAi SequencesThe BLOCK-iT? Pol II miR RNAi Subcloning Service includes a Gateway? BP and subsequent LR recombination reaction to move the miR RNAi sequence from the miR RNAi entry vector into the Gateway? destination vector of your choice. Currently there are many compatible destination vectors to choose from including lentiviral vectors and vectors with tissue specific promoters. Each destination vector has different features and benefits for increased flexibility in your experiments. We’ll help you choose the best destination vector that meets your experimental goals.
BLOCK-iT? Lentivirus ProductionFor many disease models the most desirable cell types to use, such as non-dividing or primary cells, cannot be efficiently transfected. Invitrogen’s lentiviral delivery system solves this problem by offering a powerful alternative to routine transfections. Lentiviral delivery has proven to be successful with a variety of cell types including:
Post mitotic or non-dividing cellsPrimary cellsStem cellsGrowth arrested cellsAnimal models
Once your BLOCK-iT? Pol II miR RNAi sequence is in a lentiviral destination vector, Invitrogen’s virus production team can produce either crude or concentrated lentiviral stocks in a variety of quantities and formats to meet your experimental goals.
BLOCK-iT? Pol II miR RNAi Phenotypic AssaysThe ultimate goal of gene knockdown is to observe changes in phenotype. Invitrogen’s Custom Services will work with you to design and execute a wide variety of phenotypic assays using BLOCK-iT? Pol II miR RNAi vectors.
BLOCK-iT? Pol II miR RNAi Custom ResearchInvitrogen’s team of expert scientists has years of experience working with RNAi and will work with you to design your RNAi experiments to reliably achieve your research goals. For more information on any of the BLOCK-iT? Pol II miR RNAi Custom Services listed here, please contact Invitrogen Custom Services.
Achieve transient or stable target knockdownPerform RNAi in any cell type – even hard to transfect, primary and non-diving cellsRegulate gene inhibition with inducible RNAi expressionStudy long-term gene knockdown
BLOCK-iT? Pol II miR RNAi vectors combine the benefits of traditional RNAi vectors – stable expression and the ability to use viral delivery – with capabilities for tissue-specific expression and multiple target knockdown from the same transcript. These vectors are designed to express artificial miRNAs that are engineered to have 100% homology to your target gene and result in target cleavage.
Table 1: BLOCK-iT? Pol II miR RNAi Entry VectorsmiR RNAi Entry Vector Advantages pcDNA?6.2- GW/miR Pol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors pcDNA?6.2- GW/EmGFP- miR Co-cistronic EmGFP reporter for easy tracking of miR RNAi expressionPol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors
Once your miR RNAi entry vector is generated, our RNAi services team will transfect it into mammalian cells to perform knockdown studies, chain multiple miR RNAi sequences together to knock down more than one target with the same vector, or transfer the miR RNAi sequence to another destination vector. Regardless of your vector choice, once expressed in a cell, the miR RNAi sequence induces an RNAi response resulting in knockdown of the targeted message.
Subcloning BLOCK-iT? Pol II miR RNAi SequencesThe BLOCK-iT? Pol II miR RNAi Subcloning Service includes a Gateway? BP and subsequent LR recombination reaction to move the miR RNAi sequence from the miR RNAi entry vector into the Gateway? destination vector of your choice. Currently there are many compatible destination vectors to choose from including lentiviral vectors and vectors with tissue specific promoters. Each destination vector has different features and benefits for increased flexibility in your experiments. We’ll help you choose the best destination vector that meets your experimental goals.
BLOCK-iT? Lentivirus ProductionFor many disease models the most desirable cell types to use, such as non-dividing or primary cells, cannot be efficiently transfected. Invitrogen’s lentiviral delivery system solves this problem by offering a powerful alternative to routine transfections. Lentiviral delivery has proven to be successful with a variety of cell types including:
Post mitotic or non-dividing cellsPrimary cellsStem cellsGrowth arrested cellsAnimal models
Once your BLOCK-iT? Pol II miR RNAi sequence is in a lentiviral destination vector, Invitrogen’s virus production team can produce either crude or concentrated lentiviral stocks in a variety of quantities and formats to meet your experimental goals.
BLOCK-iT? Pol II miR RNAi Phenotypic AssaysThe ultimate goal of gene knockdown is to observe changes in phenotype. Invitrogen’s Custom Services will work with you to design and execute a wide variety of phenotypic assays using BLOCK-iT? Pol II miR RNAi vectors.
BLOCK-iT? Pol II miR RNAi Custom ResearchInvitrogen’s team of expert scientists has years of experience working with RNAi and will work with you to design your RNAi experiments to reliably achieve your research goals. For more information on any of the BLOCK-iT? Pol II miR RNAi Custom Services listed here, please contact Invitrogen Custom Services.
Structure of WDR5 bound to mixed lineage Leukemia protein-1 peptide
The Mixed Lineage Leukemia protein-1 (MLL1) catalyzes histone H3 Lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (Retinoblastoma Binding Protein-5), and the Ash2L (Absent, small, homeotic discs-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine containing motif, called the “Win” or WDR5 interaction motif that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-? crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that R3765 of the MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodynamic binding experiments show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes R3765 of MLL1, which is essential for the assembly and enzymatic activity of the MLL1 core complex.
A conserved arginine containing motif crucial for the assembly and enzymatic activity of the Mixed Lineage Leukemia protein-1 core complex
The Mixed Lineage Leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD-repeat protein-5 (WDR5), retinoblastoma binding protein-5 (RbBP5), and the Absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine containing motif- called the “Win” or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5’s recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro.
Molecular Mechanism of Sequence-Directed DNA Loading and Translocation by FtsK
Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain γ. The crystal structure of three FtsKγ domains bound to 8 bp KOPS DNA demonstrates how three γ domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three γ domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.
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