9 research outputs found
c-Jun N-terminal kinase-mediated Rad18 phosphorylation facilitates PolĀ recruitment to stalled replication forks
The association of Rad18 with PolĪ· is crucial for efficient translesion synthesis and DNA damage tolerance. Rad18āPolĪ· interactions and UV tolerance depend on JNK-dependent Rad18 phosphorylation. These results provide a new mechanism by which SAPK signaling promotes genome maintenance.The E3 ubiquitin ligase Rad18 chaperones DNA polymerase Ī· (PolĪ·) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of PolĪ· with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the PolĪ·-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1ādependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damageāindependent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser ā Ala substitution at S409 was compromised for PolĪ· association and did not redistribute PolĪ· to nuclear foci or promote PolĪ·āPCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance
Phosphorylated Rad18 directs DNA Polymerase to sites of stalled replication
The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (PolĪ·) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the PolĪ·-binding motif of Rad 18 is phosphorylated by DDK. Efficient association of Rad18 with PolĪ· is dependent on DDK and is necessary for redistribution of PolĪ· to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability
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Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 lysine 27 trimethylation
Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL)1 and polysomy 21 is the most frequent somatic aneuploidy amongst all B-ALLs2. Yet, the mechanistic links between chr.21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chr.21q22 confers murine progenitor B cell self-renewal in vitro, maturation defects in vivo, and B-ALL with either BCR-ABL or CRLF2 with activated JAK2. Chr.21q22 triplication suppresses H3K27me3 in progenitor B cells and B-ALLs, and ābivalentā genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Strikingly, human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Finally, overexpression of HMGN1, a nucleosome remodeling protein encoded on chr.21q223ā5, suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo
Polymerase Ī“ promotes chromosomal rearrangements and imprecise double-strand break repair
Ā© 2020 National Academy of Sciences. All rights reserved. Recent studies have implicated DNA polymerases Īø (Pol Īø) and Ī² (Pol Ī²) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase Ī“ (Pol Ī“) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol Ī“ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol Īø, Pol Ī“ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol Ī“ promotes Alt-NHEJ in human cells at DSBs, including translocations
Platinum and PARP Inhibitor Resistance Due to Overexpression of MicroRNA-622 in BRCA1-Mutant Ovarian Cancer
High-grade serous ovarian carcinomas (HGSOCs) with BRCA1/2 mutations exhibit improved outcome and sensitivity to double-strand DNA break (DSB)-inducing agents (i.e., platinum and poly(ADP-ribose) polymerase inhibitors [PARPis]) due to an underlying defect in homologous recombination (HR). However, resistance to platinum and PARPis represents a significant barrier to the long-term survival of these patients. Although BRCA1/2-reversion mutations are a clinically validated resistance mechanism, they account for less than half of platinum-resistant BRCA1/2-mutated HGSOCs. We uncover a resistance mechanism by which a microRNA, miR-622, induces resistance to PARPis and platinum in BRCA1 mutant HGSOCs by targeting the Ku complex and restoring HR-mediated DSB repair. Physiologically, miR-622 inversely correlates with Ku expression during the cell cycle, suppressing non-homologous end-joining and facilitating HR-mediated DSB repair in S phase. Importantly, high expression of miR-622 in BRCA1-deficient HGSOCs is associated with worse outcome after platinum chemotherapy, indicating microRNA-mediated resistance through HR rescue
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PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA
Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. Here we develop a zinc-finger nuclease translocation reporter and screen for factors that modulate rearrangements in human cells. We identify UBC9 and RAD50 as suppressors and 53BP1, DDB1 and poly(ADP)ribose polymerase 3 (PARP3) as promoters of chromosomal rearrangements across human cell types. We focus on PARP3 as it is dispensable for murine viability and has druggable catalytic activity. We find that PARP3 regulates G quadruplex (G4) DNA in response to DNA damage, which suppresses repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3ā/ā cells leads to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways