Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.

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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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Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816539115 ◽

2018 ◽

Vol 115 (51) ◽

pp. E11961-E11969 ◽

Cited By ~ 14

Author(s):

Tai-Yuan Yu ◽

Michael T. Kimble ◽

Lorraine S. Symington

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Inhibitory Effects ◽

Checkpoint Signaling ◽

Synthetic Lethal ◽

Strand Breaks ◽

Damage Response ◽

End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

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RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair

10.1101/2021.04.23.440542 ◽

2021 ◽

Author(s):

Takaaki Yasuhara ◽

Reona Kato ◽

Motohiro Yamauchi ◽

Yuki Uchihara ◽

Lee Zou ◽

...

Keyword(s):

Active Regions ◽

Strand Break ◽

Double Strand Breaks ◽

Critical Component ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Chromosome Translocations ◽

Dna Double Strand Break

AbstractR-loops, consisting of ssDNA and DNA-RNA hybrids, are potentially vulnerable unless they are appropriately processed. Recent evidence suggests that R-loops can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. Yet, how the vulnerability of R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops and chromosome translocations and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end-joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

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CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks

Nature Communications ◽

10.1038/s41467-020-19502-5 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Magdalena B. Rother ◽

Stefania Pellegrino ◽

Rebecca Smith ◽

Marco Gatti ◽

Cornelia Meisenberg ◽

...

Keyword(s):

Charge Syndrome ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Multiple Levels ◽

Non Homologous End Joining ◽

And Shifts ◽

Chromatin Relaxation

AbstractChromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled ‘chromatin breathing’ upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails.

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The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks

Genes ◽

10.3390/genes12091430 ◽

2021 ◽

Vol 12 (9) ◽

pp. 1430

Author(s):

Ian Yannuzzi ◽

Margaret A. Butler ◽

Joel Fernandez ◽

Jeannine R. LaRocque

Keyword(s):

Direct Repeat ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Homology Directed Repair ◽

Dna End Resection ◽

The Impact ◽

End Resection

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.

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SIRT6 is a DNA Double-Strand Break Sensor

10.1101/765172 ◽

2019 ◽

Author(s):

Lior Onn ◽

Miguel Portillo ◽

Stefan Ilic ◽

Gal Cleitman ◽

Daniel Stein ◽

...

Keyword(s):

Dna Damage ◽

Binding Capacity ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

The Road ◽

Non Homologous End Joining

AbstractDNA double strand breaks are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure, with high affinity for double strand breaks. It relocates to sites of damage independently of signalling and known sensors and activates downstream signalling cascades for double strand break repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the Homologous Recombination and Non-Homologous End Joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as DNA damage sensor, which is critical for initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB binding capacity and DDR activation. SIRT6 activates the DDR, before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 at the top of the DDR and pave the road to dissect the contributions of distinct double strand break sensors in downstream signalling.

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The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks

BioMed Research International ◽

10.1155/2020/4834965 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Lei Zhao ◽

Chengyu Bao ◽

Yuxuan Shang ◽

Xinye He ◽

Chiyuan Ma ◽

...

Keyword(s):

Dna Repair ◽

Double Strand Breaks ◽

Ionising Radiation ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Dna Dsbs ◽

Repair Pathways

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

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Screen identifies DYRK1B network as mediator of transcription repression on damaged chromatin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002193117 ◽

2020 ◽

Vol 117 (29) ◽

pp. 17019-17030 ◽

Cited By ~ 1

Author(s):

Chao Dong ◽

Kirk L. West ◽

Xin Yi Tan ◽

Junshi Li ◽

Toyotaka Ishibashi ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Gene Silencing ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Active Chromatin ◽

Chemical Library ◽

Dsb Repair ◽

Strand Breaks ◽

Damage Response

DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B–EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.

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Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks

The Journal of Cell Biology ◽

10.1083/jcb.201109100 ◽

2012 ◽

Vol 197 (2) ◽

pp. 189-199 ◽

Cited By ~ 79

Author(s):

Maria Poulsen ◽

Claudia Lukas ◽

Jiri Lukas ◽

Simon Bekker-Jensen ◽

Niels Mailand

Keyword(s):

Deoxyribonucleic Acid ◽

Double Strand Breaks ◽

Negative Regulator ◽

Nonhomologous End Joining ◽

Genome Integrity ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Protein Recruitment

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin-dependent accumulation of repair factors at DSB sites. Instead, RNF169 functionally competed with 53BP1 and RAP80–BRCA1 for association with RNF168-modified chromatin independent of its catalytic activity, limiting the magnitude of their recruitment to DSB sites. By delaying accumulation of 53BP1 and RAP80 at damaged chromatin, RNF169 stimulated homologous recombination and restrained nonhomologous end joining, affecting cell survival after DSB infliction. Our results show that RNF169 functions in a noncanonical fashion to harness RNF168-mediated protein recruitment to DSB-containing chromatin, thereby contributing to regulation of DSB repair pathway utilization.

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Altered DNA ligase activity in human disease

Mutagenesis ◽

10.1093/mutage/gez026 ◽

2019 ◽

Vol 35 (1) ◽

pp. 51-60 ◽

Cited By ~ 3

Author(s):

Alan E Tomkinson ◽

Tasmin Naila ◽

Seema Khattri Bhandari

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Double Strand Breaks ◽

Dna Ligase ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna Ligases ◽

Ligase Iv ◽

Non Homologous End Joining

Abstract The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

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