Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells

Contribution of DNA Helicases to Genome Stability

DNA double-strand breaks (DSBs) are one of the most deleterious lesions to the cell. Even a single unrepaired DSB can lead to apoptosis, recombination, loss of heterozygosity, and cancer, thus it is essential for DSBs to be repaired. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair in mitotically dividing cells, yielding non-recombinant products. Assays that directly measure SDSA have been used to study SDSA in fruit flies and yeast, however, in humans SDSA is poorly understood and the players involved are unknown due to the lack of an SDSA assay. I have developed the first SDSA assay in human cells and present the first direct evidence for SDSA in humans. Furthermore, I report here that human BLM helicase, unlike its Drosophila ortholog is a negative regulator of SDSA. I identified RTEL1 as another negative SDSA regulator. This study provides new insights into the molecular basis of SDSA regulation and shows that BLM and RTEL1-deficient cells exhibit longer synthesis tracts which facilitates SDSA repair. To complement my studies, I utilized a DR-GFP assay to measure GC levels and engineered a crossover-gene conversion (CO-GC) assay and demonstrate here that BLM is responsible for suppression of COs in human cells despite its inhibitory effect on SDSA.Doctor of Philosoph

Regulated peristalsis into the acidic region of the _Drosophila_ larval midgut is controlled by a novel component of the Autonomic Nervous System

The underlying cellular and molecular mechanisms that regulate and coordinate critical physiological processes such as peristalsis are complex, often cryptic, and involve the integration of multiple tissues and organ systems within the organism. We have identified a completely novel component of the larval autonomic nervous system in the _Drosophila_ larval midgut that is essential for the peristaltic movement of food from the anterior midgut into the acidic region of the midgut. We have named this region the Superior Cupric Autonomic Nervous System or SCANS. Located at the junction of the anterior and the acidic portions of the midgut, the SCANS is characterized by a cluster of a novel neuro-enteroendocrine cells that we call Lettuce Head Cells, a valve, and two anterior muscular tethers to the dorsal gastric caeca. Using cell ablation and ectopic activation via expression of the _Chlamydomonas reinhardtii_ blue-light activated channelrhodopsin, we demonstrate that the SCANS and in particular the Lettuce Head Cells are both necessary and sufficient for peristalsis and perhaps serve a larger role by coordinating digestion throughout the anterior midgut with development and growth

Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells

Biodegradacja nowych polimerowych spoiw odlewniczych na przykładzie kompozycji poli(kwas akrylowy)/dekstryna

The investigations on the biodegradation process pathway of the new polymer binders for the example of water soluble composition polyacrylic acid/starch are presented in the hereby paper. Degradation was carried out in water environment and in a soil. The determination of the total oxidation biodegradation in water environment was performed under laboratory conditions in accordance with the static water test system (Zahn-Wellens method), in which the mixture undergoing biodecomposition contained inorganic nutrient, activated sludge and the polymer composition, as the only carbon and energy source. The biodecomposition progress of the polymer composition sample in water environment was estimated on the basis of the chemical oxygen demand (COD) measurements and the determination the biodegradation degree, Rt, during the test. These investigations indicated that the composition polyacrylic acid/starch constitutes the fully biodegradable material in water environment. The biodegradation degree Rt determined in the last 29th day of the test duration achieved 65%, which means that the investigated polymer composition can be considered to be fully biodegradable. During the 6 months biodegradation process of the cross-linked sample of the polymer composition in a garden soil several analysis of surface and structural changes, resulting from the sample decomposition, were performed. Those were: thermal analyses (TG-DSC), structural analyses (Raman spectroscopy) and microscopic analyses (optical microscopy, AFM).W pracy przedstawiono badania nad przebiegiem procesu biodegradacji nowych spoiw polimerowych na przykładzie wodorozpuszczalnej kompozycji poli(kwas akrylowy)/ skrobia. Degradację prowadzono w środowisku wodnym i w glebie. Oznaczenie całkowitej tlenowej biodegradacji w środowisku wodnym wykonano w warunkach laboratoryjnych zgodnie ze statycznym wodnym systemem testowym (metoda Zahna Wellensa), w którym poddana biorozkładowi mieszanina zawierała pożywkę nieorganiczną, osad czynny oraz kompozycję polimerową, jako jedyne źródło węgla i energii. Postęp biorozkładu próbki kompozycji polimerowej w środowisku wodnym oceniano na podstawie pomiarów chemicznego zapotrzebowania na tlen (ChZT) i wyznaczenia stopnia biodegradacji Rt w przygotowanych mieszaninach w trakcie trwania testu. Przeprowadzone badania biodegradacji w środowisku wodnym wykazały, że kompozycja poli(kwas akrylowy)/skrobia jest materiałem w pełni biodegradowalnym w środowisku wodnym. Stopnień biodegradacji Rt wyznaczony w ostatnim 28 dniu trwania testu osiągnął poziom 65%, co oznacza iż badaną kompozycję polimerową można uznać za w pełni biodegradowalną. Podczas trwania procesu biodegradacji próbki usieciowanej kompozycji polimerowej w glebie ogrodowej trwającego 6 miesięcy przeprowadzono analizę termiczną (TG-DSC), analizę strukturalną (spektroskopia Ramana) oraz mikroskopową (mikroskopia optyczna, AFM) zmian powierzchniowych i strukturalnych wynikających z rozkładu próbki

Pulsatile releasing platform of nanocontainers equipped with thermally responsive polymeric nanovalves

Polymer-gated reservoirs built of an ordered porous anodic aluminum oxide (AAO) platform equipped with poly( N -isopropylacrylamide) (PNIPAM) brushes grafted from the surface using atom transfer radical polymerization were fabricated and studied. The brushes of di ff erent lengths were grafted from the AAO surface with pores having diameters of 30, 50, and 80 nm, respectively. Polymer brushes served as thermally responsive valves that can immediately open and close the pores just by crossing the PNIPAM lower critical solution temperature. Calcein was used as a model fl uorescent molecule to follow a release process from the platform of the gated nanocontainers. The studied systems enable the burst release of the loaded substance, followed by di ff usion-controlled release that depends on the pores ’ diameter. These platforms are characterized by a high loading capacity in comparison to polymer fi lms and can be loaded repeatedly. Importantly, the opening/closing of the nanocontainers is reversible, and a pulsatile release can be easily realized. Such platforms combined with highly localized heating devices can fi nd potential applications in many systems where small controlled amounts of substances have to be released on demand, such as lab-on-a-chip, total analysis, and nano/micro fl uidic systems