Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

Alver, Robert C.; Chadha, Gaganmeet Singh; Gillespie, Peter J.; Blow, J. Julian

doi:10.1016/j.celrep.2017.02.042

Cited by 102 publications

(156 citation statements)

References 42 publications

(79 reference statements)

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“…The distinct colocalization patterns between RIF1 and FUS and the absence of RIF1 peptides in FUS complexes strongly suggests that FUS contributes to RT independent of RIF1. Consistent with this, while RIF1 is a well characterized suppressor of pre-RC assembly of origin firing (Alver et al, 2017), FUS enhanced pre-RC chromatin loading in early G 1 -phase cells ( Fig. 5 and Sup.…”

Section: Discussionsupporting

confidence: 78%

Fused in sarcoma regulates DNA replication timing and progression

Jia

Scalf

Tonzi

et al. 2020

Preprint

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Fused in sarcoma (FUS) encodes a low complexity RNA-binding protein with diverse roles in transcriptional activation and RNA processing.While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS has also been implicated in DNA double-strand break repair (DSBR) and genome maintenance. However, the underlying mechanisms are unknown. Here we employed quantitative proteomics, trancriptomics, and DNA copy number analysis (Sort-Seq), in conjunction with FUS -/cells to ascertain roles of FUS in genome protection. FUS-deficient cells exhibited alterations in the recruitment and retention of DSBR factors BRCA1 and 53BP1 but were not overtly sensitive to genotoxins. FUS-deficient cells exhibited reduced proliferative potential that correlated with reduced replication fork speed, diminished loading of prereplication complexes, and attenuated expression of S-phase associated genes. FUS interacted with replisome components, including lagging strand synthesis factors, but did not translocate with active replication forks. Finally, we show that FUS contributes to genome-wide control of DNA replication timing. Alterations in DNA replication initiation and timing may contribute to genome instability and functional defects in mitotically active cells harboring disease-associated FUS fusions FUS regulates pre-replication complex (pre-RC) loading and associates with DNA replication factors. Given their reduced DNA replication rate, we investigated whether FUS -/cells exhibited defects in the chromatin loading of replication licensing factors, including the origin recognition complex (ORC), CDC6, and CDT,and the MCM replicative helicase (Fragkos et al., 2015)). Mitotically arrested FUS +/+ , FUS -/-, and FUS -/-:FUS cells were released into early G 1 phase and soluble and chromatin fractions analyzed by immunoblotting. FUS-deficient cells showed normal cell progression from G 2 /M to G 1 phase and unchanged ORC loading onto chrmatin in G 1 (Fig. 5A and B). By contrast, recruitment of CDC6 and CDT1 was significantly decreased in FUS -/cells and rescued by FUS reexpression (Fig. 5B). As expected, CDC6 and CDT1-dependent loading of MCM complex was also reduced in FUS -/cells (Sup. Fig. 6). Collectively, these results revealed that FUS facilitates ORC-dependent recruitment of pre-RC factors CDC6 and CDT1 to replication origins.

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Section: Discussionsupporting

confidence: 78%

Fused in sarcoma regulates DNA replication timing and progression

Jia

Scalf

Tonzi

et al. 2020

Preprint

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“…To further understand these mechanistic differences, we assessed what role the PP1 binding motif within Rif1 has on RT control of pericentric heterochromatin in wing discs and follicle cells. Rif1 orthologs from yeasts to humans contain a PP1 binding motif, and mutation of this motif prevents Rif1 association with PP1 in multiple systems ((Davé et al 2014; Hiraga et al 2014; Mattarocci et al 2014; Sreesankar et al 2015; Alver et al 2017; Hiraga et al 2017; Sukackaite et al 2017)). We previously generated an allele of Rif1 ( Rif1 PP1 ) where the conserved SILK/RSVF PP1 interaction motif is mutated to SAAK/RASA (Munden et al 2018).…”

Section: Resultsmentioning

confidence: 99%

Rif1 functions in a tissue-specific manner to control replication timing through its PP1-binding motif

Armstrong¹,

Das

Hill³

et al. 2019

Preprint

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22Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early 23 and late replicating regions of the genome, a process known as replication timing (RT). RT changes 24 during development to ensure accurate genome duplication and maintain genome stability. To 25 understand the relative contributions that cell lineage, cell cycle, and replication initiation 26 regulators have on RT, we utilized the powerful developmental systems available in Drosophila 27 melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and 28 from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has 29 the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little 30 to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases 31 are largely independent of transcriptional differences. We also employed a genetic approach in 32 these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, 33 has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to 34 control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for 35 Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily 36 driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT 37 programs. 38

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“…Therefore, to further verify the functional involvement of MCMs in cells with perturbed chromatin compaction, we determined if DDK activity contributes to ssDNA accumulation and γH2A.X signaling. Notably, we observed a dramatic reduction in DNA damage, as evident from γH2A.X positive cells, when two different DDK inhibitors (PHA-767491 & XL413) 38–40 were added to the cells lacking SET8 (Fig. 4f and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 83%

A histone H4K20 methylation-mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing

Shoaib

Walter

Gillespie

et al. 2018

Preprint

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26The decompaction and re-establishment of chromatin organization immediately after mitosis is 27 essential for genome regulation. The mechanisms underlying chromatin structure control in 40All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/350033 doi: bioRxiv preprint first posted online Jun. 27, 2018; (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 85All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (Fig. 1a-c). Cells were 93 simultaneously labelled with methyl-14 C containing thymidine during the experiment. After 94MNase digestion, Methyl-14 C released into the supernatant was used as a measure of 95 compaction status of the cells ( Supplementary Fig. 1a). The more decompacted and accessible 96 the chromatin is, the more methyl-14 C is released in to the supernatant. Notably, the compaction 97 state of both control and siSET8 cells in mitosis were very similar (judged by the amount of 98 methyl-14 C released in the supernatant) (Fig. 1d) Supplementary Fig. 1d), it was evident that signal strength 111 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/350033 doi: bioRxiv preprint first posted online Jun. 27, 2018; 6 was higher in siSET8 cells. These data are consistent with the overall loss of chromatin 112 compaction in the absence of SET8 as also observed in the MNase assay (Fig. 1d) Supplementary Fig. 3a, b). In agreement with our MNase 128 digestion analysis (Fig. 1b), we detected a significantly lower mean FRET efficiency in siSET8 129 cells in G1 phase, but not at G2/M phases, compared to siControl cells ( Supplementary Fig. 3c, 130 d). Consistent with these results, transmission electron microscopy analysis of siControl and 131 siSET8 cells also revealed a reduction in chromatin density throughout the nucleus in SET8 132 depleted cells in G1 phase (Fig. 1g, h). Altogether these results indicate a major role for SET8 in (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/350033 doi: bioRxiv preprint first posted online Jun. 27, 2018; 7 SET8 is responsible for the methylation of histone H4 at lysine 20, which has previously been 138 implicated in chromatin compaction in in vitro assays 17 . Furthermore, H4-tail interaction with 139 an acidic patch on H2A/H2B histones on neighboring nucleosomes has also been suggested to 140 be important for maintaining ...

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Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

Cited by 102 publications

References 42 publications

Fused in sarcoma regulates DNA replication timing and progression

Fused in sarcoma regulates DNA replication timing and progression

Rif1 functions in a tissue-specific manner to control replication timing through its PP1-binding motif

A histone H4K20 methylation-mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing

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