Topoisomerase 2b induces DNA breaks to regulate human papillomavirus replication

Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2b is a type II topoisomerase that induces double strand DNA breaks at topological-associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CTCF and SMC1 cohesin proteins in complexes with TOP2b. Upon DNA cleavage a covalent intermediate DNA-TOP2b (TOP2bcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2bcc but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the HPV life cycle we investigated if TOP2b proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2b and TDP2 were substantially increased in cells with high risk HPV genomes and this correlated with high amounts of DNA breaks. Knockdown of TOP2b with shRNAs reduced DNA breaks by over 50% as determined through COMET assays. Furthermore this correlated with substantially reduced formation of repair foci such as gH2AX, pCHK1 and pSMC1 indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2b also blocked HPV genome replication. Our previous studies demonstrated that CTCF /SMC1 factors associate with HPV genomes at sites in the late regions of HPV31 and these correspond to regions that also bind TOP2b. This study identifies TOP2b as responsible for enhanced levels of DNA breaks in HPV positive cells and as a regulator of viral replication.

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Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication

mBio ◽

10.1128/mbio.00005-21 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Paul Kaminski ◽

Shiyuan Hong ◽

Takeyuki Kono ◽

Paul Hoover ◽

Laimonis Laimins

Keyword(s):

Dna Damage ◽

Human Papillomavirus ◽

Genome Replication ◽

Type Ii ◽

Torsional Stress ◽

Dna Breaks ◽

Double Strand Dna Breaks ◽

Damage Repair ◽

Topologically Associated Domains ◽

Repair Pathways

ABSTRACT Topoisomerases regulate higher-order chromatin structures through the transient breaking and religating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CCCTC-binding factor (CTCF) and SMC1 cohesin proteins in complexes with TOP2β. Upon DNA cleavage, a covalent intermediate DNA-TOP2β (TOP2βcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2βcc, but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the human papillomavirus (HPV) life cycle, we investigated whether TOP2β proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2β and TDP2 were substantially increased in cells with high-risk HPV genomes, and this correlated with large amounts of DNA breaks. Knockdown of TOP2β with short hairpin RNAs (shRNAs) reduced DNA breaks by over 50% as determined through COMET assays. Furthermore, this correlated with substantially reduced formation of repair foci such as phosphorylated H2AX (γH2AX), phosphorylated CHK1 (pCHK1), and phosphorylated SMC1 (pSMC1) indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2β also blocked HPV genome replication. Our previous studies demonstrated that CTCF/SMC1 factors associate with HPV genomes at sites in the late regions of HPV31, and these correspond to regions that also bind TOP2β. This study identifies TOP2β as responsible for enhanced levels of DNA breaks in HPV-positive cells and as a regulator of viral replication. IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate DNA damage repair pathways by inducing high numbers of DNA breaks in both viral and cellular DNAs. This activation is required for HPV genome replication. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. Our studies demonstrate that TOP2β levels are increased in HPV-positive cells and that this is required for HPV replication. Importantly, our studies further show that knockdown of TOP2β reduces the number of breaks by over 50% in HPV-positive cells and that this correlates with substantially impaired activation of DNA repair pathways. This study identifies a critical mechanism by which HPV replication is regulated by the topoisomerase TOP2β through DNA break formation.

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Double-strand DNA breaks recruit the centromeric histone CENP-A

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0908233106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15762-15767 ◽

Cited By ~ 90

Author(s):

Samantha G. Zeitlin ◽

Norman M. Baker ◽

Brian R. Chapados ◽

Evi Soutoglou ◽

Jean Y. J. Wang ◽

...

Keyword(s):

Dna Cleavage ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Strand Breaks ◽

Double Strand Dna Breaks ◽

Histone H3 Variant ◽

Radiation Induced ◽

Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

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A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

Nature Communications ◽

10.1038/s41467-019-12802-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 21

Author(s):

William H. Gittens ◽

Dominic J. Johnson ◽

Rachal M. Allison ◽

Tim J. Cooper ◽

Holly Thomas ◽

...

Keyword(s):

Dna Cleavage ◽

Genome Stability ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Strand Breaks ◽

Single Strand Breaks ◽

Human Genomes ◽

Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

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Characterization of a dam mutant of Haemophilus influenzae Rd

Microbiology ◽

10.1099/mic.0.27225-0 ◽

2004 ◽

Vol 150 (11) ◽

pp. 3773-3781 ◽

Cited By ~ 8

Author(s):

Piotr Zaleski ◽

Andrzej Piekarowicz

Keyword(s):

Haemophilus Influenzae ◽

High Sensitivity ◽

Dna Breaks ◽

Gene Encoding ◽

Phenotypic Properties ◽

Chloramphenicol Resistance ◽

Double Strand Dna Breaks ◽

Dam Methylation ◽

Dna Strands

The gene encoding Dam methyltransferase of Haemophilus influenzae was mutagenized by the insertion of a chloramphenicol-resistance cassette into the middle of the Dam coding sequence. This mutant construct was introduced into the H. influenzae chromosome by transformation and selection for CamR transformants. The authors have shown that several phenotypic properties, resistance to antibiotics, dyes and detergent as well as efficiency of transformation, depend on the Dam methylation state of the DNA. Although the major role of the methyl-directed mismatch repair (MMR) system is to repair postreplicative errors, it seems that in H. influenzae its effect is more apparent in repairing DNA damage caused by oxidative compounds. In the dam mutant treated with hydrogen peroxide, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted into single- and double-strand DNA breaks. This is shown by the increased peroxide sensitivity of the dam mutant and the finding that the sensitivity can be suppressed by a mutH mutation inactivating MMR. In the dam mutant treated with nitrofurazone the resulting damage is not converted into DNA breaks but the high sensitivity is also suppressed by a mutH mutation.

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Killing of Escherichia coli K-12 by near-ultraviolet radiation in the presence of hydrogen peroxide: Role of double-strand DNA breaks in absence of recombinational repair

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/0027-5107(80)90217-1 ◽

1980 ◽

Vol 72 (1) ◽

pp. 31-42 ◽

Cited By ~ 22

Author(s):

P.S. Hartman ◽

A. Eisenstark

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Ultraviolet Radiation ◽

Recombinational Repair ◽

Dna Breaks ◽

Near Ultraviolet ◽

Double Strand Dna Breaks ◽

K 12 ◽

Double Strand Dna

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ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

10.1101/2020.01.03.892703 ◽

2020 ◽

Author(s):

Georgia Chatzinikolaou ◽

Kalliopi Stratigi ◽

Kyriacos Agathangelou ◽

Maria Tsekrekou ◽

Evi Goulielmaki ◽

...

Keyword(s):

Dna Cleavage ◽

Genotoxic Stress ◽

Gene Promoters ◽

Dna Breaks ◽

Double Strand Dna Breaks ◽

Genome Wide ◽

Human Disorders ◽

Or Gene ◽

Type Ii Dna Topoisomerases

AbstractType II DNA Topoisomerases (TOP II) generate transient double-strand DNA breaks (DSBs) to resolve topological constraints during transcription. Using genome-wide mapping of DSBs and functional genomics approaches, we show that, in the absence of exogenous genotoxic stress, transcription leads to DSB accumulation and to the recruitment of the structure-specific ERCC1-XPF endonuclease on active gene promoters. Instead, we find that the complex is released from regulatory or gene body elements in UV-irradiated cells. Abrogation of ERCC1 or re-ligation blockage of TOP II-mediated DSBs aggravates the accumulation of transcription-associated γH2Ax and 53BP1 foci, which dissolve when TOP II-mediated DNA cleavage is inhibited. An in vivo biotinylation tagging strategy coupled to a high-throughput proteomics approach reveals that ERCC1-XPF interacts with TOP IIβ and the CTCF/cohesin complex, which co-localize with the heterodimer on DSBs. Together; our findings provide a rational explanation for the remarkable clinical heterogeneity seen in human disorders with ERCC1-XPF defects.

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125I Decay in Microorganisms: A Prokaryotic Model System for Examining the Role of Unrepaired Double-Strand DNA Breaks in Cell Killing

Chromosome Damage and Repair ◽

10.1007/978-1-4684-7956-0_25 ◽

1981 ◽

pp. 193-197

Author(s):

Robert E. Krisch

Keyword(s):

Model System ◽

Cell Killing ◽

Dna Breaks ◽

Double Strand Dna Breaks ◽

Double Strand Dna

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Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

10.1101/362558 ◽

2018 ◽

Cited By ~ 16

Author(s):

Hannah A. Grunwald ◽

Valentino M. Gantz ◽

Gunnar Poplawski ◽

Xiang-ru S. Xu ◽

Ethan Bier ◽

...

Keyword(s):

Dna Cleavage ◽

Mendelian Inheritance ◽

Early Embryo ◽

Gene Drive ◽

Dna Breaks ◽

Homology Directed Repair ◽

Double Strand Dna Breaks ◽

Repair Mechanisms ◽

Island Communities ◽

Random Assortment

AbstractA gene drive biases the transmission of a particular allele of a gene such that it is inherited at a greater frequency than by random assortment. Recently, a highly efficient gene drive was developed in insects, which leverages the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, this powerful system would enable the rapid assembly of genotypes that involve multiple genes (e.g., to model multigenic human diseases). Such complex genetic models are currently precluded by time, cost, and a requirement for a large number of animals to obtain a few individuals of the desired genotype. However, the efficiency of a CRISPR/Cas9 gene drive system in mammals has not yet been determined. Here, we utilize an active genetic “CopyCat” element embedded in the mouse Tyrosinase gene to detect genotype conversions after Cas9 activity in the embryo and in the germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and is therefore highly mutagenic, these breaks are not resolved by homology directed repair. However, when Cas9 expression is limited to the developing female germline, resulting double strand breaks are resolved by homology directed repair that copies the CopyCat allele from the donor to the receiver chromosome and leads to its super-Mendelian inheritance. These results demonstrate that the CRISPR/Cas9 gene drive mechanism can be implemented to simplify complex genetic crosses in laboratory mice and also contribute valuable data to the ongoing debate about applications to combat invasive rodent populations in island communities.

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A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

10.1101/530667 ◽

2019 ◽

Cited By ~ 2

Author(s):

William Gittens ◽

Dominic J. Johnson ◽

Rachal M. Allison ◽

Tim J. Cooper ◽

Holly Thomas ◽

...

Keyword(s):

Dna Cleavage ◽

Genome Stability ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Strand Breaks ◽

Single Strand Breaks ◽

Human Genomes ◽

Nucleotide Resolution

AbstractDNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage—distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

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