Chromatin structure, not DNA sequence specificity, is the primary determinant of topoisomerase II sites of action in vivo

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.

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The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7522 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7522-7530 ◽

Cited By ~ 19

Author(s):

A Udvardy ◽

P Schedl

Keyword(s):

Heat Shock ◽

Chromatin Structure ◽

Topoisomerase Ii ◽

Chromatin Condensation ◽

Intergenic Spacer ◽

Domain Boundaries ◽

Shock Locus ◽

Sites Of Action ◽

Putative Domain

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.

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Mapping yeast mitotic 5’ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo

10.1101/2021.02.27.433206 ◽

2021 ◽

Author(s):

Dominic Bazzano ◽

Stephanie Lomonaco ◽

Thomas E. Wilson

Keyword(s):

Double Strand Breaks ◽

Sequence Specificity ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Endonucleolytic Cleavage ◽

Step Model ◽

Local Sequence ◽

Exact Counting ◽

Exact Position

AbstractResection of the 5’-terminated strand at DNA double strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Biochemical and genetic studies have led to a multi-step model of DSB resection in which endonucleolytic cleavage mediated by Mre11 in partnership with Sae2 is coupled with exonucleolytic cleavage mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods commonly used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with next-generation sequencing in which unique molecular identifiers allow exact counting of cleaved 5’ ends at base pair resolution. Mutant strains, including exo1Δ, mre11-H125N, exo1Δ and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60 to 70 bp from the DSB end whose exact position depended on local sequence and tracked an apparent motif. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB and near the Exo1 pause point and where it was much less restrained. These results provide detailed in vivo support of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

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DNA Double-Strand Breaks Are a Critical Regulator of Fear Memory Reconsolidation

International Journal of Molecular Sciences ◽

10.3390/ijms21238995 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8995

Author(s):

Shaghayegh Navabpour ◽

Jessie Rogers ◽

Taylor McFadden ◽

Timothy J. Jarome

Keyword(s):

Transcriptional Control ◽

Topoisomerase Ii ◽

Fear Memory ◽

Double Strand Breaks ◽

Memory Reconsolidation ◽

Long Term Memory ◽

Epigenetic Mark ◽

Dna Double Strand Breaks ◽

Strand Breaks

Numerous studies have shown that following retrieval, a previously consolidated memory requires increased transcriptional regulation in order to be reconsolidated. Previously, it was reported that histone H3 lysine-4 trimethylation (H3K4me3), a marker of active transcription, is increased in the hippocampus after the retrieval of contextual fear memory. However, it is currently unknown how this epigenetic mark is regulated during the reconsolidation process. Furthermore, though recent evidence suggests that neuronal activity triggers DNA double-strand breaks (DSBs) in some early-response genes, it is currently unknown if DSBs contribute to the reconsolidation of a memory following retrieval. Here, using chromatin immunoprecipitation (ChIP) analyses, we report a significant overlap between DSBs and H3K4me3 in area CA1 of the hippocampus during the reconsolidation process. We found an increase in phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker of DSB, in the Npas4, but not c-fos, promoter region 5 min after retrieval, which correlated with increased H3K4me3 levels, suggesting that the two epigenetic marks may work in concert during the reconsolidation process. Consistent with this, in vivo siRNA-mediated knockdown of topoisomerase II β, the enzyme responsible for DSB, prior to retrieval, reduced Npas4 promoter-specific H2A.XpS139 and H3K4me3 levels and impaired long-term memory, indicating an indispensable role of DSBs in the memory reconsolidation process. Collectively, our data propose a novel mechanism for memory reconsolidation through increases in epigenetic-mediated transcriptional control via DNA double-strand breaks.

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The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7522-7530.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7522-7530

Author(s):

A Udvardy ◽

P Schedl

Keyword(s):

Heat Shock ◽

Chromatin Structure ◽

Topoisomerase Ii ◽

Chromatin Condensation ◽

Intergenic Spacer ◽

Domain Boundaries ◽

Shock Locus ◽

Sites Of Action ◽

Putative Domain

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.

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ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

10.1101/2019.12.18.881409 ◽

2019 ◽

Cited By ~ 2

Author(s):

Jacob Paiano ◽

Wei Wu ◽

Shintaro Yamada ◽

Nicholas Sciascia ◽

Elsa Callen ◽

...

Keyword(s):

Meiotic Recombination ◽

Homologous Chromosome ◽

Direct Detection ◽

Double Strand Breaks ◽

Sequence Specificity ◽

Dna Sequence Specificity ◽

Strand Breaks ◽

Genome Wide ◽

Meiotic Dsbs

AbstractMeiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11. The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations (“hotspots”) are determined by the DNA sequence specificity of PRDM9. Here, we demonstrate the first direct detection of meiotic DSBs and resection in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum lengths of resected DNA, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate (“SPO11-RI”) present at all hotspots. The SPO11-bound intermediate is dependent on PRDM9 and caps the 3’ resected end during engagement with the homologous template. We propose that SPO11-RI is generated because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm−/− spermatocytes, SPO11-RI is reduced while unresected DNA-bound SPO11 accumulate because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.

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Focused Genetic Recombination of Bacteriophage T4 Initiated by Double-Strand Breaks

Genetics ◽

10.1093/genetics/162.2.543 ◽

2002 ◽

Vol 162 (2) ◽

pp. 543-556

Author(s):

Victor Shcherbakov ◽

Igor Granovsky ◽

Lidiya Plugina ◽

Tamara Shcherbakova ◽

Svetlana Sizova ◽

...

Keyword(s):

Genetic Recombination ◽

Bacteriophage T4 ◽

Proximal Part ◽

Coupling Model ◽

Strand Break ◽

Double Strand Breaks ◽

Strand Breaks ◽

The Right ◽

Frequency Distance

Abstract A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCΔ strain of bacteriophage T4 was developed. The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene. Under segC+ conditions, the ets1 behaves as a recombination hotspot. Crosses of the ets1 against rII markers located to the left and to the right of ets1 gave similar results, thus demonstrating the equal and symmetrical initiation of recombination by either part of the broken chromosome. Frequency/distance relationships were studied in a series of two- and three-factor crosses with other rIIB and rIIA mutants (all segC+) separated from ets1 by 12-2100 bp. The observed relationships were readily interpretable in terms of the modified splice/patch coupling model. The advantages of this localized or focused recombination over that distributed along the chromosome, as a model for studying the recombination-replication pathway in T4 in vivo, are discussed.

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Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

DNA Repair ◽

10.1016/j.dnarep.2006.12.002 ◽

2007 ◽

Vol 6 (5) ◽

pp. 639-648 ◽

Cited By ~ 19

Author(s):

Yukitaka Katsura ◽

Shigeru Sasaki ◽

Masanori Sato ◽

Kiyoshi Yamaoka ◽

Kazumi Suzukawa ◽

...

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks

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Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

The Journal of Cell Biology ◽

10.1083/jcb.200608077 ◽

2007 ◽

Vol 177 (2) ◽

pp. 219-229 ◽

Cited By ~ 256

Author(s):

Naoya Uematsu ◽

Eric Weterings ◽

Ken-ichi Yano ◽

Keiko Morotomi-Yano ◽

Burkhard Jakob ◽

...

Keyword(s):

Kinase Activity ◽

Laser System ◽

Double Strand Breaks ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

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