Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks

Kim, Hyun Suk; Nickoloff, Jac A.; Wu, Yuehan; Williamson, Elizabeth A.; Sidhu, G. S.; Reinert, Brian L.; Jaiswal, Aruna S.; Srinivasan, Gayathri; Patel, Bhavesh; Kong, Kimi; Burma, Sandeep; Lee, Suk Hee; Hromas, Robert

doi:10.1074/jbc.m116.758235

Cited by 32 publications

(46 citation statements)

References 43 publications

(178 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…EXO1 knockdown showed the strongest effect on CHK1 phosphorylation. Moreover, in HEK-293 cells EXO1 knockdown significantly reduced ssDNA formation after 2h of HU treatment [39]. We did not observe a reduction in RPA2-S4/S8 phosphorylation upon knockdown of any of the potential resection enzymes.…”

Section: Ddk Likely Promotes Fork Processing By Regulating the Activimentioning

confidence: 62%

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

Sasi

Coquel

Lin

et al. 2017

Preprint

View full text Add to dashboard Cite

Abstract:CDC7-DBF4 kinase (DDK) is required to initiate DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has also been reported to have diverse and sometimes conflicting roles in the replication checkpoint response in various organisms but the underlying mechanisms are far from settled. Here 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 we show that EXO1 is also required for nascent strand degradation following exposure to HU, raising the possibility that DDK regulates EXO1 directly. Thus, 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.Keywords: DNA replication checkpoint/ replication fork/ fork resection/ fork restart/ DDK peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/207266 doi: bioRxiv preprint first posted online 3 DDK is a two-subunit kinase that is essential to initiate DNA replication at individual replication origins by phosphorylating and activating the MCM2-7 replicative helicase. The regulatory subunit DBF4 binds to CDC7 and is required for its kinase activity [1]. DDK also has roles in replication checkpoint signaling that are less well understood [1]. In budding yeast, DDK is a target of the checkpoint effector kinase Rad53 that is activated following replication stress [2]. Rad53-mediated phosphorylation of Dbf4 modestly reduces DDK activity [2] and also inhibits late origin firing [3,4], but there is also evidence that DDK is required for the complete activation of Rad53 kinase [5]. In fission yeast, DDK subunits Hsk1 and Dfp1 are phosphorylated upon HU treatment by the checkpoint kinase Cds1, orthologous to budding yeast Rad53 and mammalian CHK2 [6][7][8]. Deletion of either Cds1 or its activating protein Mrc1 partially rescues the temperature sensitivity of hsk1-1312 mutants suggesting that Cds1 (like Rad53) inhibits DDK activity [8,9]. Cds1 activation in response to HU, however, was reduced significantly in hsk1(ts) strains at restrictive temperature and the cell cycle arrest was also aberrant as seen by an increased population of 'cut' cells (indicative of mitosis without complete DNA replication) [8,10]. These paradoxical results in yeast could be explained by a negative feedback loop where DDK first helps to initiate replication checkpoint activation and then the checkpoint pathway subsequently alters DDK activity to inhibit late origin firing.Initial studies in human cells showed that the replication checkpoint inhibits DDK activity, presumably to inhibit origin firing [11,...

show abstract

Section: Ddk Likely Promotes Fork Processing By Regulating the Activimentioning

confidence: 62%

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

Sasi

Coquel

Lin

et al. 2017

Preprint

View full text Add to dashboard Cite

show abstract

“…Herein, MRX generates the substrate for the recruitment of Exo1 or Sgs1-Dna2. This resembles the situation in mammalian DNA replication fork repair wherein the endonuclease EEPD1 nicks the stalled fork and loads Exo1 onto that structure (44).…”

Section: Dna Nick Processing By Exo1 and Sgs1-dna2mentioning

confidence: 86%

A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1–Dna2 in long-range DNA end resection

Wang

Daley

Kwon

et al. 2018

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.

show abstract

“…The QTL in 1B of both populations displayed a large PVE, 15.1% in Pop1 and 11.25% in Pop2. According to the BLAST results, QGZn.cimmyt.1B_P1 appears to overlap with a gene that codes a protein belonging to the endo/exonuclease/phosphatase domain, which function is associated with DNA binding and repair during DNA replication (Wu et al, 2015 ; Kim et al, 2017 ). The QTL in chromosome 1B from Pop2 was located in a region where genes code for proteins with unknown function.…”

Section: Discussionmentioning

confidence: 99%

QTL Mapping of Grain Zn and Fe Concentrations in Two Hexaploid Wheat RIL Populations with Ample Transgressive Segregation

et al. 2017

View full text Add to dashboard Cite

More than 50% of undernourished children live in Asia and more than 25% live in Africa. Coupled with an inadequate food supply, mineral deficiencies are widespread in these populations; particularly zinc (Zn) and iron (Fe) deficiencies that lead to retarded growth, adverse effects on both the immune system and an individual's cognitive abilities. Biofortification is one solution aimed at reducing the incidence of these deficiencies. To efficiently breed a biofortified wheat variety, it is important to generate knowledge of the genomic regions associated with grain Zn (GZn) and Fe (GFe) concentration. This allows for the introgression of favorable alleles into elite germplasm. In this study we evaluated two bi-parental populations of 188 recombinant inbred lines (RILs) displaying a significant range of transgressive segregation for GZn and GFe during three crop cycles in CIMMYT, Mexico. Parents of the RILs were derived from Triticum spelta L. and synthetic hexaploid wheat crosses. QTL analysis identified a number of significant QTL with a region denominated as QGZn.cimmyt-7B_1P2 on chromosome 7B explaining the largest (32.7%) proportion of phenotypic variance (PVE) for GZn and leading to an average additive effect of −1.3. The QTL with the largest average additive effect for GFe (−0.161) was found on chromosome 4A (QGFe.cimmyt-4A_P2), with 21.14% of the PVE. The region QGZn.cimmyt-7B_1P2 co-localized closest to the region QGZn.cimmyt-7B_1P1 in a consensus map built from the linkage maps of both populations. Pleiotropic or tightly linked QTL were also found on chromosome 3B, however of minor effects and PVE between 4.3 and 10.9%. Further efforts are required to utilize the QTL information in marker assisted backcrossing schemes for wheat biofortification. A strategy to follow is to intercross the transgressive individuals from both populations and then utilize them as sources in biofortification breeding pipelines.

show abstract

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks

Cited by 32 publications

References 43 publications

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1–Dna2 in long-range DNA end resection

QTL Mapping of Grain Zn and Fe Concentrations in Two Hexaploid Wheat RIL Populations with Ample Transgressive Segregation

Contact Info

Product

Resources

About