Note: Dynamic behavior of DNA topoisomerase IIbeta in response to DNA double-strand breaks

Note: Dynamic behavior of DNA topoisomerase IIbeta in response to DNA double-strand breaks

(doi: 10.1038/s41598-018-28690-6)

DNA topoisomerase II (Topo II) -plays important roles in various cellular processes, such as replication, transcription, and chromosome segregation.

DNA topology problems may also occur during DNA repair, the possible involvement of Topo II in this process remains to be fully investigated.

rapid recruitment of EGFP-tagged Topo IIβ to the DSB site.

Topo IIβ is highly mobile in the nucleus. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 reduced the Topo IIβ mobility and thereby prevented Topo IIβ recruitment to DSBs. Furthermore, Topo IIβ knockout cells exhibited increased sensitivity to bleomycin and decreased DSB repair mediated by homologous recombination (HR), implicating the role of Topo Iiβ in HR-mediated DSB repair.

DNA topoisomerase II (Topo II) is an ATP-dependent enzyme that resolves DNA topological problems, such as supercoiling and catenation.

DNA replication, transcription, and chromosome condensation and segregation, all of which can give rise to

topological constraints of chromosomal DNA.

an ATPase domain in the N-terminus, a central catalytic domain, a C-terminal domain.

most of which fall into two classes, namely Topo II poisons and Topo II catalytic inhibitors. Topo II poisons, such as etoposide, halt the Topo II catalytic reaction cycle during formation of a covalent Topo II-DNA complex, which is readily converted into a DSB in living cells. While Topo II poisons produce DSBs, Topo II catalytic inhibitors block the catalytic cycle without a marked increase in DSB production.

In mammals, there are two Topo II isozymes, Topo IIα and Topo IIβ, which share striking sequence homology

with one another in their N-terminal ATPase and central catalytic domains but differ in their C-terminal

domains.

they exhibit characteristic expression patterns and play distinct roles in cellular processes. Topo IIα is highly expressed in proliferating cells with peak expression at late S and G2/M phases of the cell cycle. Topo IIα primarily functions in the resolution of DNA topology problems that arise during DNA replication and mitosis. Topo IIβ is expressed in both dividing and non-dividing cells, and its function is largely dispensable for cell cycle progression.

implicated Topo IIβ in the transcription of subsets of genes, such as developmentally-controlled genes and hormone-regulated genes.

ATM is activated by DSB induction and in turn undergoes autophosphorylation at DSB sites. Activated ATM influences DSB repair by phosphorylating multiple nuclear proteins.

NHEJ is thus active throughout the cell cycle, and DNA-dependent protein kinase (DNA-PK) plays a critical role in this mechanism. NHEJ is thus active throughout the cell cycle, and DNA-dependent protein kinase (DNA-PK) plays a critical role in this mechanism. DNA-PKcs belongs to the PI3KK family, and its activation manifests as autophosphorylation.

chromatin remodeling is also important in DSB repair. histone deacetylase (HDAC) members are recruited to DSB sites and are involved in chromatin remodeling during DSB repair. Poly(ADP-ribose) polymerase-1 (PARP-1) is rapidly recruited to DSB sites and modifies histones and chromatin proteins with poly(ADP-ribose) to facilitate DSB repair.

Topo IIβ forms a large complex with DNA-PKcs, Ku, and PARP-1 on the promoters of estrogen-, insulin-, and glucocorticoid-responsive genes.

However, it remains unclear how Topo II itself responds to DSB induction. Topo II participates in various cellular processes associated with DNA topology problems, such as replication and mitosis, limited information is available regarding the involvement of Topo II in DSB repair, which may also require the control of DNA topology.

Topo IIβ with DSB repair factors in transcriptional control, we aimed to explore the response of Topo IIβ to DSB induction.

recruitment of Topo IIβ to laser-induced DSB sites. Topo IIβ recruitment to DSB sites was partially abrogated by inhibitors for PARP-1 and HDAC, but, contrary to our assumption, it did not require DNA-PKcs or ATM. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 prevented Topo IIβ recruitment to DSBs by reducing Topo IIβ mobility.

nucleus using a pulsed UVA laser (349 nm) for induction of localized DNA damage. a pulsed UVA laser can achieve high energy and thereby produce both DSBs and single strand breaks (SSBs).

we observed punctate foci of endogenous Topo IIβ in the irradiated nucleus. Topo IIβ foci were colocalized with those of phosphorylated DNA-PKcs, indicating that endogenous Topo IIβ was recruited to damaged sites.

endogenous Topo IIβ and Ku70 were retained as foci in detergent-extracted cells and colocalized with each other.

Human Topo IIβ fused to EGFP was transiently expressed in HeLa cells, and

DNA damage was induced by microirradiation with a pulsed UVA laser.

EGFP-Topo IIβ was recruited to the damaged sites immediately after laser microirradiation. Recruitment of EGFP-Topo IIβ was transient, and fluorescent signals for EGFP-Topo IIβ foci gradually became faint and then marginally detectable after 20 min.

Topo IIβ was recruited to DSB sites, owing to the observed colocalization of Topo IIβ with phosphorylated DNA-PKcs and Ku70 at laser-induced foci.

Line-scanning of the nucleus with a 405 nm laser produces SSBs, but not DSBs, due to its insufficient energy. Hoechst 33342 serves as a photosensitizer that enables the production of DSBs by 405 nm laser line scanning. First, we confirmed the production of SSBs after 405 nm laser scanning without Hoechst 33342 by examining the

localization of EGFP-XRCC1, which is known to be recruited to SSB sites.

indicating the production of SSBs by 405 nm laser scanning.

When cells were pretreated with Hoechst 33342, EGFP-Topo IIβ was recruited to the scanned area, supporting the notion that Topo IIβ is recruited to DSB sites.

Recruitment of Topo IIβ to DSB sites is independent of ATM and DNA-PKcs -- Topo IIβ recruitment to DSB sites is under the control of ATM and DNA-PKcs, both of which are protein

kinases of the PI3KK family that play important roles in DSB signaling and repair.

microirradiation of ATM-deficient and -proficient cells followed by coimmunostaining for endogenous Topo IIβ and phosphorylated ATM.

These observations indicate that ATM activation is dispensable for Topo IIβ recruitment. these results indicate

that Topo IIβ recruitment to DSB sites is not dependent on ATM or DNA-PKcs.

cellular responses to DSB induction are modulated by posttranslational modifications of histones and chromatin-associated proteins around DSB sites. Topo IIβ recruitment to DSB sites, we analyzed the effects of inhibitors of PARP-1 and HDAC, both of which play important roles in DSB-induced chromatin modifications.

these observations indicate that the catalytic activities of PARP-1 and HDAC are required for Topo IIβ recruitment, presumably through chromatin remodeling around DSB sites.

The Topo II catalytic inhibitors ICRF-187 and ICRF-193 halt the conformational transitions of Topo II at the closed clamp step. Topo II poisons, treatment with these inhibitors does not cause a significant increase in DSBs. ICRF-187 and ICRF-193, we next investigated the impact of conformational restraints of Topo IIβ on recruitment

to DSB sites.

For comparison to a different class of Topo II catalytic inhibitors, we next used merbarone, which inhibits DNA cleavage by Topo II without affecting DNA-Topo II association. We found that merbarone did not interfere with Topo IIβ recruitment to DSB sites.

Previous studies have reported that amino acid substitutions alter the enzymatic properties and ICRF sensitivity

of Topo II. Similarly, rat Topo IIβ harboring an L178F substitution in its ATPase domain is insensitive to the ICRF compounds.

In the absence of ICRF-187, all mutant proteins were recruited to DSB sites in a similar manner to that seen for the

wild-type protein. This indicates that neither ATPase nor Topo II catalytic activity was required for Topo IIβ recruitment to DSB sites.

These observations support a causal relationship between ICRF treatment and the suppression of Topo IIβ recruitment to DSB sites.

Topo IIα demonstrated that ICRF-187 decreases the nuclear mobility of Topo IIα and thereby inhibits Topo IIα functions in mitosis.

that ICRF-187 may also affect Topo IIβ mobility.

In the absence of ICRF-187, EGFP-Topo IIβ was highly mobile in the nucleus as evident from the rapid recovery of the EGFP signals in the photobleached area. Quantification of FRAP data demonstrated that virtually all EGFP-Topo IIβ molecules were mobile, since there was nearly 100% fluorescence recovery after photobleaching.

presence of ICRF-187, fluorescence recovery in the photobleached area was barely detectable in fluorescence

microscopy. Approximately 20% of EGFP-Topo IIβ was estimated to be mobile from quantification analysis, suggesting that a large fraction of EGFP-Topo IIβ was tethered to chromatin. ICRF-187 to reduced Topo IIβ mobility.

The nuclear distribution of Ku70 was not affected by ICRF-187; Ku70 was retained at DSB sites and was washed out from undamaged area after detergent treatment. Taken together with the observations from the FRAP analysis, we concluded that ICRF-187 caused a tight association of Topo IIβ with chromatin and reduced the mobility of Topo IIβ, which could account for the failure of Topo IIβ recruitment to DSB sites.

To compare with the effects of Topo II catalytic inhibitors, we next tested etoposide, a Topo II poison. the nuclear mobility of EGFP-Topo IIβ was slightly reduced by etoposide, presumably because a fraction of EGFP-Topo IIβ was tethered to chromatin DNA.

Given the recruitment of Topo IIβ to DSB sites, we next sought to gain insight into the role of Topo IIβ in DSB repair. previously generated human Topo IIβ knockout cells by gene targeting in the Nalm-6 cell line.

Topo IIβ knockout cells were more sensitive to bleomycin than wild-type cells, which was particularly evident at high doses. Because HR plays a larger role in the repair of severe DSBs than NHEJ, we next examined the effect of Topo IIβ gene knockout on HR-mediated DSB repair. The DR-GFP reporter cassette for the HR assay  was knocked-in to the HPRT locus on the X chromosome of Topo IIβ knockout and wild-type cells.

When a DSB in the DR-GFP reporter is repaired by HR, this yields GFP-positive cells. When a DSB in the DR-GFP reporter is repaired by HR, this yields GFP-positive cells.

limited information is available on the dynamic behavior of Topo IIβ, particularly in relation to DSB induction. we investigated the response of Topo IIβ to DSB induction and its physiological significance. Live cell-imaging showed rapid and transient recruitment of EGFP-Topo IIβ to DSB sites.

the high mobility of EGFP-Topo IIβ in the nucleus.

The Topo II catalytic inhibitors ICRF-187 and ICRF-193 decreased the nuclear mobility of EGFP-Topo IIβ and prevented the recruitment of EGFP-Topo IIβ to DSB sites.

Since ICRF-187 and ICRF-193 are known to trap the homodimeric Topo II protein as a closed clamp on a DNA strand,  we speculated that these inhibitors caused the tethering of Topo IIβ to chromatin DNA and thereby interfered with the translocation of Topo IIβ to DSB sites.

visible foci do not necessarily reflect the initial recruitment step of DSB responses. subsequent signal amplification and protein clustering surrounding the damaged sites are more important for the formation of visible foci. Topo IIβ binds to DSBs at relatively low abundance and with fast kinetics.

raise new questions that need to be addressed in future research. First, the mechanism for Topo IIβ recruitment to damaged sites remains an open question. Among various forms of DNA damage, we inferred that Topo IIβ is recruited to DSBs, because the recruitment of Topo IIβ to 405 nm laser scanning sites required presensitization with Hoechst 33342.

we examined the possible participation of two major regulatory factors, namely ATM and DNA-PKcs, in Topo IIβ recruitment.

Topo IIβ recruitment to DSB sites was indistinguishable between ATM-proficient and -deficient cells. Topo IIβ associates with DNA-PKcs on the promoter regions of several hormone-responsive genes. the association between Topo IIβ and DNA-PKcs is crucial for transcriptional control, this was not the case for the recruitment of Topo IIβ to DSB sites, since Topo IIβ recruitment was observed in cells lacking DNA-PKcs.

these observations demonstrated that Topo IIβ recruitment to DSBs is independent of ATM or DNA-PKcs. In contrast, Topo IIβ recruitment was affected with inhibitors for PARP-1 and HDAC, both of which contribute to alterations in chromatin structure around DSB sites.

on identifying a molecular event that is required for Topo IIβ recruitment upon DSB induction.

We thus favor the view that the greater sensitivity of Topo IIβ knockout cells to high-dose bleomycin implies the role of Topo IIβ in HR-mediated DSB repair. This idea will be validated by detailed mechanistic analysis in the future.

Our observation on the reduced activity of HR-mediated DSB repair in Topo IIβ-deficient cells led to another open question of how Topo IIβ is involved in this process.

We hypothesize an auxiliary role of Topo IIβ in HR, presumably through indirect mechanisms, rather than direct involvement in HR. Topo IIβ may have a facilitative role in the modulation of topological constraints of DNA strands imposed during the process of HR. protective role of Topo IIβ in DNA topology around DSBs to ensure

efficient DSB repair.

Topo IIβ deficiency may cause certain structural alterations around DSB sites and ultimately manifest as a reduced HR activity. impact of Topo IIβ deficiency on NHEJ in comparison to HR is also important for understanding the significance of Topo IIβ in DSB repair. sheds light on novel aspects of Topo IIβ function and provides a basis for further research into the mechanism by which Topo IIβ knockout affects HR-mediated DSB repair.

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