DNA Breaks and End Resection Measured Genome-wide by End Sequencing
Andrés CanelaSriram SridharanNicholas SciasciaAnthony TubbsPaul S. MeltzerBarry P. SleckmanAndré Nussenzweig
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Nuclease
The reassociation kinetics of Escherichia coli DNA were measured by S1 nuclease resistance and hydroxyapatite binding. While the reaction assayed by hydroxyapatite displays second order kinetics, the S1 nuclease measurements follow a non-second order from, as previously reported by Morrow (Ph.D. Dissertation, Stanford University. 1974). Much of the reaction measured with S1 nuclease occurs between single stranded regions of fragments already bearing duplex structures from previous collisions, and between such regions and totally free single strands. Experimental determinations indicate that the nucleation rate of single stranded regions on fragments also containing duplexes is inhibited by an average factor of 2 to 4.
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Isolated cell nuclei were incubated with nucleases, and then the chromatin was extracted with a low-salt buffer. When degradation of the nuclear chromatin DNase I or micrococcal nuclease is intensified, solubilization of the deoxyribonucleoprotein (DNP) in low-salt buffer at first increases, reaching a maximum in the case of hydrolysis of 2-4% of the nuclear DNA, but after intensive treatment with nucleases, it decreases sharply. Soluble fragmented chromatin is aggregated during treatment with DNase I. The addition of exogenous products of nuclease treatment of isolated nuclei to a preparation of gelatinous chromatin induces its aggregation. Pretreatment of nuclear chromatin with RNase prevents the solubilization of DNP by solutions with low ionic strength. Certain experimental data obtained using rigorous nuclease treatment are discussed; for their interpretation it is necessary to consider the effect of aggregation of fragmented chromatin by products of its nuclease degradation.
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Gene density
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This study presents a novel approach for mapping global chromatin interactions using S1 nuclease, a sequence-agnostic enzyme. We develop and outline a protocol that leverages S1 nuclease's ability to effectively introduce breaks into both open and closed chromatin regions, allowing for comprehensive profiling of chromatin properties. Our S1 Hi-C method enables the preparation of high-quality Hi-C libraries, marking a significant advancement over previously established DNase I Hi-C protocols. Moreover, S1 nuclease's capability to fragment chromatin to mono-nucleosomes suggests the potential for mapping the three-dimensional organization of the genome at high resolution. This methodology holds promise for an improved understanding of chromatin state-dependent activities and may facilitate the development of new genomic methods.
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The studies reported here demonstrate that ATP may be used in lieu of EDTA to inhibit nuclease digestion of DNA and chromatin. Because ATP is a milder chelator than EDTA and is a biochemical common to the cellular microenvironment in vivo, critical studies of cellular processes that require native structure to be maintained are more feasible without the presence of strong chelators. During the digestion of chromatin into its components by nuclease treatment, ATP assures the retention of nucleoprotein compaction, particularly for large to intermediate-sized oligosomes (2400bp-1000bp in length). ATP used at a concentration of 3.3 mM appears to be somewhat better than EDTA, 1.0 mM, for minimizing degradation of nucleasetreated chromatin. However, termination of nuclease digestion of chromatin and minimization of further degradation by the addition of ATP to a concentration of 1.0 mM was almost equivalent to the addition of EDTA to a concentration of 1.0 mM. Slightly more degradation was observed for the latter condition. In addition, ATP can be used to inhibit endogenous nuclease activity when specific restriction enzymes are needed. Standard low ionic strength DNP, deoxyribonucleoprotein, and DNA electrophoresis of proteinized and deproteinized chromatin oligomers, respectively, indicated that ATP effectively inhibits staphylococcal nuclease. Low ionic strength nucleoprotein electrophoresis to resolve staphylococcal nuclease-digested chromatin indicates that as little as 10 -4 M EDTA can promote structural unfolding resulting in changes in apparent mobilities for chromatin oligomers 250 and 600 bp in length. Comparative digestion of chromatin with staphlococcal nuclease followed by reaction termination by ATP or EDTA showed that this observation was not merely the result of degradation due to inefficiency of ATP enzyme inhibition.
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Abstract Inherent nanometer-sized features and molecular recognition properties make DNA a useful material in constructing nanoscale objects, with alluring applications in biosensing and drug delivery. However, DNA can be easily degraded by nucleases present in biological fluids, posing a considerable roadblock to realizing the full potential of DNA nanotechnology for biomedical applications. Here we investigated the nuclease resistance and biostability of the multi-stranded motif called paranemic crossover (PX) DNA and discovered a remarkable and previously unreported resistance to nucleases. We show that PX DNA has more than an order of magnitude increased resistance to degradation by DNase I, serum, and urine compared to double stranded DNA. We further demonstrate that the degradation resistance decreases monotonically as DNA crossovers are removed from the structure, suggesting that frequent DNA crossovers disrupt either the binding or catalysis of nucleases or both. Further, we show using mouse and human cell lines that PX DNA does not affect cell proliferation or interfere with biological processes such as myogenesis. These results have important implications for building DNA nanostructures with enhanced biostability, either by adopting PX-based architectures or by carefully engineering crossovers. We contend that such crossover-dependent nuclease resistance could potentially be used to add “tunable biostability” to the many features of DNA nanotechnology.
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Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.
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The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.
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The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.
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The characteristics of the postirradiation degradation of chromatin in thymocytes in vivo were compared with the features of chromatin fragmentation in isolated thymocyte nuclei in vitro by endogenous chromatin-bound nucleases. Nuclease which degrades chromatin produces in vivo fragments of nucleosomal size; the double-strand breaks appear as the result of the accumulation of single-strand breaks with 3'-OH ends; the nuclease is inhibited by Zn2+ and DTNB and its activity is depressed by cycloheximide pretreatment. In experiments on in vitro degradation of chromatin in isolated thymocyte nuclei similar properties were observed for the Ca, Mg-dependent, but not for acid nuclease. The results bring further evidence of the involvement of an enzyme of the Ca, Mg-dependent nuclease-type in chromatin degradation in irradiated thymocytes.
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Thymocyte
Micrococcal nuclease
Fragmentation
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