Structure of the human ATM kinase and mechanism of Nbs1 binding

Kinase Domain ◽

Atm Kinase ◽

Strand Breaks ◽

Mrn Complex ◽

Biochemical Assays ◽

Hydrophobic Cleft ◽

Yeast Homolog

DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and ultimately cancer. Central to the sensing of DSBs are ATM (Ataxia telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, and the MRN (Mre11-Rad50-Nbs1) protein complex that activates ATM. How the MRN complex recruits and activates ATM kinase is poorly understood. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM kinase, and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped autoinhibited structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning the N and C lobes along with relieving the blockage of the substrate-binding site. We show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these interactions with mutagenesis and biochemical assays.

SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining

Nucleic Acids Research ◽

10.1093/nar/gkab051 ◽

2021 ◽

Author(s):

Ekaterina Akimova ◽

Franz Josef Gassner ◽

Maria Schubert ◽

Stefan Rebhandl ◽

Claudia Arzt ◽

...

Keyword(s):

Genome Instability ◽

Hek293 Cells ◽

Dna Breaks ◽

End Joining ◽

Strand Breaks ◽

Intracellular Dntp ◽

Samhd1 Expression ◽

Nucleotide Insertions

Abstract Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.

MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts

Nature Communications ◽

10.1038/s41467-019-12271-w ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Emily Yun-Chia Chang ◽

Shuhe Tsai ◽

Maria J. Aristizabal ◽

James P. Wells ◽

Yan Coulombe ◽

...

Keyword(s):

Dna Replication ◽

Fanconi Anemia ◽

Genome Instability ◽

Double Strand Breaks ◽

Strand Breaks ◽

Mrn Complex ◽

Genome Wide ◽

A Genome ◽

Dna Replication Stress

Abstract Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors including the MRE11-RAD50-NBS1 (MRN) complex. While MRN has been shown to promote R-loops at DNA double-strand breaks, we show that it suppresses R-loops and associated DNA damage at transcription–replication conflicts. This occurs through a non-nucleolytic function of MRE11 that is important for R-loop suppression by the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms at transcription–replication conflicts.

Faculty Opinions recommendation of MRN complex function in the repair of chromosomal Rag-mediated DNA double-strand breaks.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1158234.618359 ◽

2009 ◽

Author(s):

David Schatz

Keyword(s):

Complex Function ◽

Double Strand Breaks ◽

Strand Breaks ◽

Mrn Complex

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706392114 ◽

2017 ◽

Vol 114 (29) ◽

pp. 7665-7670 ◽

Cited By ~ 25

Author(s):

Chun-Chin Chen ◽

Elizabeth M. Kass ◽

Wei-Feng Yen ◽

Thomas Ludwig ◽

Mary Ellen Moynahan ◽

...

Keyword(s):

Synthetic Lethality ◽

Critical Role ◽

Nonhomologous End Joining ◽

End Joining ◽

Atm Kinase ◽

Strand Breaks ◽

Homology Directed Repair ◽

Dna Damage Signaling ◽

Mutant Cells

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1–53BP1 antagonism and that its HDR function can become critical in certain contexts.

Emerging Technologies for Genome-Wide Profiling of DNA Breakage

Frontiers in Genetics ◽

10.3389/fgene.2020.610386 ◽

2021 ◽

Vol 11 ◽

Author(s):

Matthew J. Rybin ◽

Melina Ramic ◽

Natalie R. Ricciardi ◽

Philipp Kapranov ◽

Claes Wahlestedt ◽

...

Keyword(s):

Genome Instability ◽

Single Nucleotide ◽

Strand Breaks ◽

Single Strand Breaks ◽

Genome Wide ◽

A Genome ◽

Wide Scale ◽

Nucleotide Resolution ◽

Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

Dynamics of RecA-mediated repair of replication-dependent DNA breaks

The Journal of Cell Biology ◽

10.1083/jcb.201803020 ◽

2018 ◽

Vol 217 (7) ◽

pp. 2299-2307 ◽

Cited By ~ 12

Author(s):

Vincent Amarh ◽

Martin A. White ◽

David R.F. Leach

Keyword(s):

Temporal Dynamics ◽

Double Strand Breaks ◽

Dna Breaks ◽

Chromosomal Locus ◽

Chromosomal Replication ◽

Strand Breaks ◽

Sister Chromosome ◽

Temporal Efficiency

Chromosomal replication is the major source of spontaneous DNA double-strand breaks (DSBs) in living cells. Repair of these DSBs is essential for cell viability, and accuracy of repair is critical to avoid chromosomal rearrangements. Repair of replication-dependent DSBs occurs primarily by homologous recombination with a sister chromosome. However, this reaction has never been visualized at a defined chromosomal locus, so little is known about its spatial or temporal dynamics. Repair of a replication-independent DSB generated in Escherichia coli by a rare-cutting endonuclease leads to the formation of a bundle of RecA filaments. In this study, we show that in contrast, repair of a replication-dependent DSB involves a transient RecA focus localized in the central region of the cell in which the DNA is replicated. The recombining loci remain centrally located with restricted movement before segregating with little extension to the period of postreplicative sister-chromosome cohesion. The spatial and temporal efficiency of this reaction is remarkable.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

Nucleic Acids Research ◽

10.1093/nar/gkq1098 ◽

2010 ◽

Vol 39 (5) ◽

pp. 1692-1702 ◽

Cited By ~ 43

Author(s):

Derek J. Richard ◽

Kienan Savage ◽

Emma Bolderson ◽

Liza Cubeddu ◽

Sairei So ◽

...

Keyword(s):

Double Strand Breaks ◽

Strand Breaks ◽

Mrn Complex

Parp3 promotes long-range end joining in murine cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801591115 ◽

2018 ◽

Vol 115 (40) ◽

pp. 10076-10081 ◽

Cited By ~ 6

Author(s):

Jacob V. Layer ◽

J. Patrick Cleary ◽

Alexander J. Brown ◽

Kristen E. Stevenson ◽

Sara N. Morrow ◽

...

Keyword(s):

Embryonic Stem ◽

Cell Types ◽

Strand Break ◽

Nonhomologous End Joining ◽

End Joining ◽

Strand Breaks ◽

Class Switch

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class–switch recombination in primary B cells, and inversions in tail fibroblasts that generateEml4–Alkfusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing ofEml4–Alkjunctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

Nature Communications ◽

10.1038/s41467-020-18506-5 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Andrea M. Kaminski ◽

John M. Pryor ◽

Dale A. Ramsden ◽

Thomas A. Kunkel ◽

Lars C. Pedersen ◽

...

Keyword(s):

Strand Break ◽

Nonhomologous End Joining ◽

End Joining ◽

Strand Breaks ◽

Template Strand ◽

Single Strand Breaks ◽

Dna Double Strand Break ◽

Break Site

Abstract Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

Heterochromatin and the DNA damage response: the need to relaxThis paper is one of a selection of papers in a Special Issue entitled 31st Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o10-113 ◽

2011 ◽

Vol 89 (1) ◽

pp. 45-60 ◽

Cited By ~ 72

Author(s):

Kendra L. Cann ◽

Graham Dellaire

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Chromatin Structure ◽

Peer Review Process ◽

Double Strand Breaks ◽

Atm Kinase ◽

Strand Breaks ◽

Damage Response ◽

Chromosomal Mutations

Higher order chromatin structure has an impact on all nuclear functions, including the DNA damage response. Over the past several years, it has become increasingly clear that heterochromatin and euchromatin represent separate entities with respect to both damage sensitivity and repair. The chromatin compaction present in heterochromatin helps to protect this DNA from damage; however, when lesions do occur, the compaction restricts the ability of DNA damage response proteins to access the site, as evidenced by its ability to block the expansion of H2AX phosphorylation. As such, DNA damage in heterochromatin is refractory to repair, which requires the surrounding chromatin structure to be decondensed. In the case of DNA double-strand breaks, this relaxation is at least partially mediated by the ATM kinase phosphorylating and inhibiting the function of the transcriptional repressor KAP1. This review will focus on the functions of KAP1 and other proteins involved in the maintenance or restriction of heterochromatin, including HP1 and TIP60, in the DNA damage response. As heterochromatin is important for maintaining genomic stability, cells must maintain a delicate balance between allowing repair factors access to these regions and ensuring that these regions retain their organization to prevent increased DNA damage and chromosomal mutations.