Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

Thakar, Tanay; Leung, Wendy; Nicolae, Claudia M.; Clements, Kristen E.; Shen, Binghui; Bielinsky, Anja‐Katrin; Moldovan, George‐Lucian

doi:10.1038/s41467-020-16096-w

Cited by 105 publications

(161 citation statements)

References 74 publications

(121 reference statements)

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“…In addition to damaged-induced ubiquitylation, PCNA was shown to be mono-ubiquitylated during replication of undamaged sperm chromatin ( Leach and Michael, 2005 ). A recent report suggests that this is also likely the case in human cells ( Thakar et al., 2020 ), and our data generated during replication of undamaged plasmids agree with these findings. Although the significance of damage-independent PCNA mono-ubiquitylation remains unclear, RFWD3 depletion did not affect it, suggesting independent mechanisms for inducing PCNA ubiquitylation at the fork during unstressed replication versus at ssDNA gaps upon DNA damage and RFWD3 regulating only the latter of the two.…”

Section: Discussionsupporting

confidence: 92%

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

Gallina¹,

Hendriks²,

Hoffmann³

et al. 2021

Molecular Cell

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Summary Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

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Section: Discussionsupporting

confidence: 92%

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

Gallina¹,

Hendriks²,

Hoffmann³

et al. 2021

Molecular Cell

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“…Consistent with the Ki67 staining results, our previous study showed that there was a significant reduction of PCNA levels in MnTE-2-PyP-treated prostate cancer cells with or without radiation [95]. PCNA is a key component in cell cycle progression, especially in S phase for precise DNA replication and DNA damage repair [192][193][194][195]. acetylation on K403 of G6PD will prevent the formation of the functional dimer and reduces G6PD activity, but acetylation on K76 and K294 of 6PGD will increase 6PGD activity [196][197][198][199].…”

Section: Mnte-2-pyp Inhibits Gr Activity By Depleting Nadph Poolsupporting

confidence: 77%

Mechanisms by Which MnTE-2-PyP Suppresses Prostate Cancer Cell Growth

Zhu¹

2019

Free Radical Biology and Medicine

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MECHANISMS BY WHICH MNTE-2-PYP SUPPRESSES PROSTATE CANCER CELL GROWTH Prostate cancer patients are often treated with radiotherapy. MnTE-2-PyP, is a superoxide dismutase (SOD) mimic and a known radioprotector of normal tissues. Our recent work demonstrates that MnTE-2-PyP also inhibits prostate cancer progression with radiotherapy; however, the mechanisms remain unclear. In this thesis, we identified that MnTE-2-PyP-induced intracellular H2O2 levels are critical in inhibiting growth of prostate cancer cells. We found that MnTE-2-PyP induced protein oxidations in PC3 cells and one major group of oxidized protein targets were involved in energy metabolism. The oxidative phosphorylation rates were significantly enhanced in both PC3 and LNCaP cells with MnTE-2-PyP treatment, but mitochondrial membrane potential was unaffected. In addition, MnTE-2-PyP significantly increased NAD(P)+/NAD(P)H ratios in PC3 and LNCaP cells in a dose-dependent manner, which was mainly due to a reduction of cellular NAD(P)H pool. Correspondingly, we observed a significant decrease of activity in glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), which are major cellular NADPH producing enzymes in pentose phosphate pathway. A decrease of GSH/GSSG ratios were confirmed in MnTE-2-PyP-treated prostate cancer cells, which may result from the decreased glutathione reductase (GR) activity due to NADPH depletion. We also identified the oxidation of Ser/Thr protein phosphatase 1 beta catalytic subunit (PP1CB), and a decrease of PP1CB activity in MnTE-2-PyP-treated prostate cancer cells. One key protein, pRB, regulates cell cycle progression that is downstream target of PP1CB was hypophosphorylated in MnTE-2-PyP-treated prostate cancer cells. Significant increase of Ki67-negative populations were observed in both PC3 and LNCaP cells but overall cell cycle progression was not altered, which indicates xv TABLE OF CONTENTS

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“…Therefore, we measured the levels of chromatin-bound PCNA-Ub as an indicator of the relative number of active replication forks. Although baseline PCNA-Ub is significantly lower than in HCT116 cancer cells, it is detectable under unperturbed conditions 34 . Because of this, we were able to determine that MCM10 mutants had significantly lower PCNA-Ub (Fig.…”

Section: Resultsmentioning

confidence: 88%

Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening

et al. 2021

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Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.

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Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

Cited by 105 publications

References 74 publications

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

Mechanisms by Which MnTE-2-PyP Suppresses Prostate Cancer Cell Growth

Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening

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