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DDK Has a Primary Role in Processing Stalled Replication Forks to Initiate Downstream Checkpoint Signaling.

ABSTRACT: CDC7-DBF4 kinase (DDK) initiates DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has diverse and apparently conflicting roles in the replication checkpoint response in various organisms, but the underlying mechanisms are far from settled. We show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1 and phosphorylates EXO1 in vitro. EXO1 is also required for nascent strand degradation following exposure to HU, so DDK might regulate EXO1 directly. Lastly, sublethal DDK inhibition causes various mitotic abnormalities, which is consistent with a checkpoint deficiency. In summary, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.

SUBMITTER: Sasi NK

PROVIDER: S-EPMC6111017 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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DDK Has a Primary Role in Processing Stalled Replication Forks to Initiate Downstream Checkpoint Signaling.

Sasi Nanda Kumar NK Coquel Flavie F Lin Yea-Lih YL MacKeigan Jeffrey P JP Pasero Philippe P Weinreich Michael M

Neoplasia (New York, N.Y.) 20180826 10

CDC7-DBF4 kinase (DDK) initiates DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has diverse and apparently conflicting roles in the replication checkpoint response in various organisms, but the underlying mechanisms are far from settled. We show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M c ...[more]

PMID: 30157471

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Project description:Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that Atrx(FoxG1Cre) forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1- and Brn2-positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks, limiting genomic damage, and facilitating late-born neuron production.

Dataset Information

DDK Has a Primary Role in Processing Stalled Replication Forks to Initiate Downstream Checkpoint Signaling.

Publications

DDK Has a Primary Role in Processing Stalled Replication Forks to Initiate Downstream Checkpoint Signaling.

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