Faculty Opinions recommendation of Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex.

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

Download Full-text

Double-strand breaks trigger MRX- and Mec1-dependent, but Tel1-independent, checkpoint activation

FEMS Yeast Research ◽

10.1111/j.1567-1364.2006.00076.x ◽

2006 ◽

Vol 6 (5) ◽

pp. 836-847 ◽

Cited By ~ 13

Author(s):

Muriel Grenon ◽

Christine P. Magill ◽

Noel F. Lowndes ◽

Stephen P. Jackson

Keyword(s):

Double Strand Breaks ◽

Checkpoint Activation ◽

Strand Breaks

Download Full-text

Repair Of DNA Double-Strand Breaks By Non-Homologous End-Joining Is Independent Of Cisplatin-Induced Checkpoint Activation And Downstream Damage Response Pathways In A Non-Small Cell Lung Cancer Cell Culture Model

10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a5064 ◽

2011 ◽

Author(s):

Catherine R. Sears ◽

John J. Turchi

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Cell Culture Model ◽

Lung Cancer Cell ◽

End Joining ◽

Checkpoint Activation ◽

Strand Breaks ◽

Culture Model ◽

Damage Response ◽

Non Homologous End Joining

Download Full-text

UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair

Nature Communications ◽

10.1038/s41467-021-24201-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Greg H. P. Ngo ◽

Julia W. Grimstead ◽

Duncan M. Baird

Keyword(s):

Active Regions ◽

Strand Break ◽

Dna Helicase ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Checkpoint Activation ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Dna Resection

AbstractDNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.

Download Full-text

Dpb4 promotes resection of DNA double-strand breaks and checkpoint activation by acting in two different protein complexes

Nature Communications ◽

10.1038/s41467-021-25090-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Erika Casari ◽

Elisa Gobbini ◽

Marco Gnugnoli ◽

Marco Mangiagalli ◽

Michela Clerici ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Polymerase ◽

Protein Complexes ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Checkpoint Activation ◽

Strand Breaks ◽

Checkpoint Protein ◽

Histone Fold ◽

Histone Fold Domain

AbstractBudding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase ε (Pol ε) holoenzyme. In Saccharomyces cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol ε complex. Here, we show that Dpb4 plays two functions in sensing and processing DNA double-strand breaks (DSBs). Dpb4 promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. Persistence of both Isw2 and Rad9 at DSBs is enhanced by the A62S mutation that is located in the Dpb4 histone fold domain and increases Dpb4 association at DSBs. Thus, Dpb4 exerts two distinct functions at DSBs depending on its interactors.

Download Full-text

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks

Cellular and Molecular Life Sciences ◽

10.1007/s00018-011-0875-3 ◽

2011 ◽

Vol 69 (9) ◽

pp. 1447-1473 ◽

Cited By ~ 70

Author(s):

Karen Finn ◽

Noel Francis Lowndes ◽

Muriel Grenon

Keyword(s):

Dna Damage ◽

Double Strand Breaks ◽

Dna Damage Checkpoint ◽

Checkpoint Activation ◽

Strand Breaks ◽

Eukaryotic Dna

Download Full-text

LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation

Nucleic Acids Research ◽

10.1093/nar/gkz263 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6236-6249 ◽

Cited By ~ 9

Author(s):

Kirk L West ◽

Jessica L Kelliher ◽

Zhanzhan Xu ◽

Liwei An ◽

Megan R Reed ◽

...

Keyword(s):

Dna Damage ◽

Double Strand Breaks ◽

Parp Inhibition ◽

Current Evidence ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

End Joining ◽

Checkpoint Activation ◽

Strand Breaks ◽

Non Homologous End Joining

Abstract The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.

Download Full-text

TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus of Tetrahymena

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0469 ◽

2006 ◽

Vol 17 (12) ◽

pp. 5185-5197 ◽

Cited By ~ 12

Author(s):

J. Sebastian Yakisich ◽

Pamela Y. Sandoval ◽

Tara L. Morrison ◽

Geoffrey M. Kapler

Keyword(s):

Dna Damage ◽

Genotoxic Stress ◽

S Phase ◽

Double Strand Breaks ◽

Wild Type ◽

Checkpoint Activation ◽

Checkpoint Response ◽

Strand Breaks ◽

Origin Binding Protein ◽

S Phase Checkpoint

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.

Download Full-text