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Review
. 2019 Oct 30;10(11):868.
doi: 10.3390/genes10110868.

Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA

Suguru Morimoto  1 , Masataka Tsuda  2 , Heeyoun Bunch  3 , Hiroyuki Sasanuma  4 , Caroline Austin  5 , Shunichi Takeda  6
Affiliations
Review

Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA

Suguru Morimoto et al. Genes (Basel). .

Abstract

Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this 'abortive catalysis' can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones.

Keywords: BRCA1; BRCA2; Topoisomerase II; breast cancer; cell cycle; estrogen; genotoxicity; transcription.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Release of replication blockage by breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2). DNA replication forks often stall at both damaged nucleotides (A) and single-strand breaks (B) on template strands (Step 1). Homology-directed repair (HDR) proteins including BRCA1 and BRCA2 initiate strand invasion of the 3′ end of nascent DNA strands into the intact sister chromatid (Step 3) followed by DNA synthesis (Step 4, green arrow). This pathway allows the fork to move past the damaged nucleotides (A) and single-strand breaks (B) (Step 5).
Figure 2
Figure 2
Abortive catalysis of topoisomerases causes DNA cleavage. (A) TOP1 cleaves one strand of a DNA double helix and religates the nick (Step 1 and 2). Topoisomerase I (TOP1) is covalently associated with 3′ end generating a TOP1 cleavage complex (TOP1cc shown in Step 2). TOP1 occasionally fails to religate, generating stable TOP1ccs (Step 3). 3′ TOP1 adducts are removed by the proteasome and TDP enzymes (Step 4) followed by religation of single-strand breaks (step 5). (B) A topoisomerase II (TOP2) homodimer generates a gated double-strand breaks (DSB) called a TOP2 cleavage complex (TOP2cc), where TOP2 covalently associates with 5′ end of DSB (Step 2). When TOP2 fails to religate TOP2ccs called abortive catalysis of TOP2 (Step 3), religation of the DSB is carried out by the removal of 5′ TOP2 adducts (Step 4) preceding nonhomologous end joining (NHEJ) (Step 5). (C) Two major sources of spontaneously arising DSBs, the blockage of replication forks leading to replication fork collapse (left) and abortive catalysis of TOP2 leading to the generation of stalled TOP2ccs (right).
Figure 3
Figure 3
Removal of 5′ TOP2 adducts. (A) DSBs are repaired by HDR or NHEJ. DSBs occurring during DNA replication are repaired exclusively by HDR (Figure 1). The choice between HDR and NHEJ is determined by DSB resection, the generation of 3′ single-strand tails, which inhibit NHEJ and stimulate HDR. (B) DSB resection is carried out by the coordinated action of the MRE11, EXO1, and DNA2 nucleases. Endonucleolytic cleavage of 5′ strands by MRE11 near DSB ends removals 5′ TOP2 adducts together with oligonucleotides. (C) NHEJ ligates ‘clean’ DSBs having 5′-phosphate and 3′-hydroxyl moieties but not DSBs bearing blocking adducts. 5′ TOP2 adducts are partially digested by the proteasome generating DSBs carrying 5′ oligopeptides. 5′ oligopeptides are removed by tyrosyl-DNA phosphodiesterase 2 (TDP2) (Left) or MRE11 (Right). Resulting ‘clean’ DSBs are repaired by NHEJ, which is initiated by binding of the KU70/KU80 heterodimer to DSB ends.
Figure 4
Figure 4
Model of topoisomerase IIB (TOP2B)-mediated DSB and DNA damage response signaling during RNA polymerase II (Pol II) pause release in stimulus-inducible protein-coding genes in humans. Top in the uninduced state of transcription, Pol II is paused between +25 and +100 from the transcription start site. The pausing is attributed to different elements including pausing-stabilizing transcription factors, the +1 nucleosome, and DNA structure and torsion. Positive supercoiling ahead of Pol II may require the function of TOP2B. Middle, transcription activation induced by various stimuli activates TOP2B to resolve DNA torsion in the promoter and gene body. Bottom, in this process, DSB could be formed from abortive catalysis of TOP2B, which occurs frequently in some genes. This may be responsible for DNA damage response signaling that has been observed in a number of stimulus-inducible genes in humans [59,66,70].
Figure 5
Figure 5
The roles of BRCA1 and BRCA2 in genome maintenance. (A) They play a critical role in the repair of DSBs formed during DNA replication by promoting strand exchange between two sister chromatids as shown in Figure 1. (B) BRCA1 and BRCA2 reduce the accumulation of transcription-associated R loop. (C) BRCA1 and BRCA2 promote the repair of stalled TOP2ccs as shown in Figure 3C.
Figure 6
Figure 6
Estrogen-induced TOP2B catalysis causes the formation of stalled TOP2Bccs presumably at transcriptional regulatory sequences. (Step 1) Estrogen receptors (ER) bind at their target genes. (Step 2) TOP2B catalysis at transcriptional regulatory sequences, and (Step 3) estrogens upregulate the transcription of ER-target genes. (Step 4) TOP2B undergoes abortive catalysis generating stalled TOP2Bccs. (Step 5) From the resulting lethal DSBs TOP2B adducts are removed by the coordinated action of BRCA1, BRCA2, and Mre11. (Step 6) NHEJ seals DSBs leading a cycle of TOP2B-mediated catalyses (Step 2).
Figure 7
Figure 7
TOP2B catalysis and the formation of TOP2ccs affect transcriptional responses to estrogens. TOP2B catalysis changes the three-dimensional structure of ER-target genes such as C-MYC and induces their transcription. DSB-repair-proficient normal cells are capable of very quickly repairing stalled TOP2Bccs. The loss of BRCA1 or BRCA2 protein causes accumulation of unrepaired DSBs, which may upregulate the transcriptional response of oncogenes to estrogens.
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