Inhibition of the DNA Damage Pathway by a Telomere-Binding Protein

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

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Monitoring S. pombe genome stress by visualizing end-binding protein Ku

10.1101/2020.06.12.149054 ◽

2020 ◽

Author(s):

Chance Jones ◽

Susan L Forsburg

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Protein Complex ◽

Genome Stability ◽

Binding Protein ◽

Dna Lesions ◽

Live Cells ◽

Dna Damage Checkpoint ◽

Unique Pattern ◽

Ku Protein

AbstractStudies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

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PARP-1–dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713912115 ◽

2018 ◽

Vol 115 (8) ◽

pp. E1759-E1768 ◽

Cited By ~ 14

Author(s):

Jung-Kuei Chen ◽

Wen-Ling Lin ◽

Zhang Chen ◽

Hung-wen Liu

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Strand Break ◽

Double Strand Break ◽

Dsb Repair ◽

Active Involvement ◽

Parp 1

Maintenance of genome integrity is critical for both faithful propagation of genetic information and prevention of mutagenesis induced by various DNA damage events. Here we report cold-inducible RNA-binding protein (CIRBP) as a newly identified key regulator in DNA double-strand break (DSB) repair. On DNA damage, CIRBP temporarily accumulates at the damaged regions and is poly(ADP ribosyl)ated by poly(ADP ribose) polymerase-1 (PARP-1). Its dissociation from the sites of damage may depend on its phosphorylation status as mediated by phosphatidylinositol 3-kinase-related kinases. In the absence of CIRBP, cells showed reduced γH2AX, Rad51, and 53BP1 foci formation. Moreover, CIRBP-depleted cells exhibited impaired homologous recombination, impaired nonhomologous end-joining, increased micronuclei formation, and higher sensitivity to gamma irradiation, demonstrating the active involvement of CIRBP in DSB repair. Furthermore, CIRBP depleted cells exhibited defects in DNA damage-induced chromatin association of the MRN complex (Mre11, Rad50, and NBS1) and ATM kinase. CIRBP depletion also reduced phosphorylation of a variety of ATM substrate proteins and thus impaired the DNA damage response. Taken together, these results reveal a previously unrecognized role for CIRBP in DSB repair.

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Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication

Journal of Cell Science ◽

10.1242/jcs.114.10.1861 ◽

2001 ◽

Vol 114 (10) ◽

pp. 1861-1866 ◽

Cited By ~ 1

Author(s):

J. Postberg ◽

S.A. Juranek ◽

S. Feiler ◽

H. Kortwig ◽

F. Jonsson ◽

...

Keyword(s):

Cell Cycle ◽

Nuclear Localization ◽

Nuclear Matrix ◽

Binding Protein ◽

Specific Interaction ◽

Eukaryotic Cells ◽

Dna Molecules ◽

Telomere Binding Protein ◽

The Matrix

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8)individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

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Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

Nature Communications ◽

10.1038/s41467-020-19674-0 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Alessandro Cicconi ◽

Rekha Rai ◽

Xuexue Xiong ◽

Cayla Broton ◽

Amer Al-Hiyasat ◽

...

Keyword(s):

Dna Damage ◽

Replication Fork ◽

Clinical Feature ◽

Replication Forks ◽

Primary Microcephaly ◽

Dna Damage And Repair ◽

Homology Directed Repair ◽

Replication Fork Progression ◽

Telomere Binding Protein ◽

Telomere Replication

AbstractTelomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1–TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

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The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein

Genes & Development ◽

10.1101/gad.14.14.1777 ◽

2000 ◽

Vol 14 (14) ◽

pp. 1777-1788 ◽

Cited By ~ 1

Author(s):

Haiyan Qi ◽

Virginia A. Zakian

Keyword(s):

Dna Polymerase ◽

Binding Protein ◽

Point Mutations ◽

Single Strand ◽

Catalytic Subunit ◽

Full Length ◽

Temperature Sensitive ◽

Dna Polymerase Α ◽

Telomere Binding Protein

Saccharomyces telomeres consist of ∼350 bp of C1-3A/TG1-3 DNA. Most of this ∼350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C1-3A strand is degraded to generate a long single strand TG1-3 tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG1-3DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase α, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 orPOL1 that reduced the Cdc13p–Pol1p interaction resulted in telomerase mediated telomere lengthening. Over–expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG1-3 strand lengthening by telomerase and its interaction with Pol1p promotes C1-3A strand resynthesis by DNA polymerase α.

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