PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks

Poly adenosine diphosphate (ADP)–ribosylation (PARylation) by poly ADP-ribose (PAR) polymerases (PARPs) is an early response to DNA double-strand breaks (DSBs). In this paper, we exploit Dictyostelium discoideum to uncover a novel role for PARylation in regulating nonhomologous end joining (NHEJ). PARylation occurred at single-strand breaks, and two PARPs, Adprt1b and Adprt2, were required for resistance to this kind of DNA damage. In contrast, although Adprt1b was dispensable for PARylation at DSBs, Adprt1a and, to a lesser extent, Adprt2 were required for this event. Disruption of adprt2 had a subtle impact on the ability of cells to perform NHEJ. However, disruption of adprt1a decreased the ability of cells to perform end joining with a concomitant increase in homologous recombination. PAR-dependent regulation of NHEJ was achieved through promoting recruitment and/or retention of Ku at DSBs. Furthermore, a PAR interaction motif in Ku70 was required for this regulation and efficient NHEJ. These data illustrate that PARylation at DSBs promotes NHEJ through recruitment or retention of repair factors at sites of DNA damage.

Download Full-text

A game of musical chairs: Pro- and anti-resection factors compete for TOPBP1 binding after DNA damage

The Journal of Cell Biology ◽

10.1083/jcb.201701038 ◽

2017 ◽

Vol 216 (3) ◽

pp. 535-537 ◽

Cited By ~ 1

Author(s):

Kenji Shimada ◽

Susan M. Gasser

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Binding Sites ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Pathway Regulation

DNA double strand breaks (DSBs) are generally repaired through nonhomologous end joining or homologous recombination. In this issue, Liu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607031) report that the conserved scaffold protein TOPBP1Dpb11 provides binding sites for both pro- and anti-resection factors at DSBs, providing insights into repair pathway regulation.

Download Full-text

DNA Damage-Inducible and RAD52-Independent Repair of DNA Double-Strand Breaks in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/154.3.1085 ◽

2000 ◽

Vol 154 (3) ◽

pp. 1085-1099 ◽

Cited By ~ 1

Author(s):

Carol Wood Moore ◽

Judith McKoy ◽

Michelle Dardalhon ◽

Darline Davermann ◽

Marcia Martinez ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Homologous Recombination ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Γ Irradiation ◽

Strand Breaks ◽

Mutant Strains

Abstract Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 γ irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following γ irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by γ irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after γ irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.

Download Full-text

Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

Molecular and Cellular Biology ◽

10.1128/mcb.25.3.896-906.2005 ◽

2005 ◽

Vol 25 (3) ◽

pp. 896-906 ◽

Cited By ~ 71

Author(s):

James M. Daley ◽

Thomas E. Wilson

Keyword(s):

Gc Content ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Single Strand Annealing ◽

Primary Determinant ◽

Overhang Length ◽

Strand Annealing

ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

Download Full-text

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504868112 ◽

2015 ◽

Vol 112 (24) ◽

pp. 7507-7512 ◽

Cited By ~ 63

Author(s):

Ozge Gursoy-Yuzugullu ◽

Marina K. Ayrapetov ◽

Brendan D. Price

Keyword(s):

Dna Damage ◽

Double Strand Breaks ◽

Histone H4 ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Nucleosome Dynamics ◽

Chromatin Template ◽

End Resection

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.

Download Full-text

Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

Nucleic Acids Research ◽

10.1093/nar/gkz680 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9410-9422 ◽

Cited By ~ 1

Author(s):

Andrea M Kaminski ◽

Kishore K Chiruvella ◽

Dale A Ramsden ◽

Thomas A Kunkel ◽

Katarzyna Bebenek ◽

...

Keyword(s):

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Steady State Kinetics ◽

Ligase Iv ◽

Template Dna ◽

Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

Download Full-text

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):

Chromosomal Rearrangements ◽

Strand Break ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

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.

Download Full-text

Inactivation of Ku-Mediated End Joining Suppresses mec1Δ Lethality by Depleting the Ribonucleotide Reductase Inhibitor Sml1 through a Pathway Controlled by Tel1 Kinase and the Mre11 Complex

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10652-10664.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10652-10664 ◽

Cited By ~ 13

Author(s):

Yves Corda ◽

Sang Eun Lee ◽

Sylvine Guillot ◽

André Walther ◽

Julie Sollier ◽

...

Keyword(s):

Dna Damage ◽

Ribonucleotide Reductase ◽

Reductase Inhibitor ◽

Nonhomologous End Joining ◽

Dna Damage Checkpoint ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Ribonucleotide Reductase Inhibitor ◽

Kinase Cascade

ABSTRACT RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Δ, but not rad53Δ, lethality. We show that suppression of mec1Δ lethality is not due to Ku−-associated telomeric defects but rather results from the inability of Ku− cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Δ lethality is also suppressed by lif1Δ, which like yku70Δ and yku80Δ, prevents nonhomologous end joining. The viability of yku70Δ mec1Δ and yku80Δ mec1Δ cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.

Download Full-text

Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ

The Journal of Cell Biology ◽

10.1083/jcb.200810132 ◽

2009 ◽

Vol 184 (4) ◽

pp. 515-526 ◽

Cited By ~ 30

Author(s):

Susan J. Hsiao ◽

Susan Smith

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Adenosine Diphosphate ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

End Joining ◽

Strand Breaks ◽

Ligase Iv ◽

G2 Phase ◽

Tankyrase 1

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

Download Full-text

Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

Journal of Experimental Medicine ◽

10.1084/jem.20081915 ◽

2008 ◽

Vol 205 (13) ◽

pp. 3031-3040 ◽

Cited By ~ 34

Author(s):

Likun Du ◽

Mirjam van der Burg ◽

Sergey W. Popov ◽

Ashwin Kotnis ◽

Jacques J.M. van Dongen ◽

...

Keyword(s):

Class Switch Recombination ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Igg Subclass ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Immunoglobulin Class ◽

Class Switch ◽

Human B Cells

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Sμ–Sα junctions could be amplified from all patients tested and were characterized by a complete lack of “direct” end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Sμ–Sγ junctions could only be amplified from one patient who carries “hypomorphic” mutations. Although these Sμ–Sγ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sγ–Sγ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

Download Full-text