Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

Singh, Mayank; Hunt, Clayton R.; Pandita, Raj K.; Kumar, Rakesh; Yang, Chuanchuan; Horikoshi, Nobuo; Bachoo, Robert; Serag, Sara; Story, Michael D.; Shay, Jerry W.; Powell, Simon N.; Gupta, A.; Jeffery, Jessie; Pandita, Shruti; Chen, Benjamin P.C.; Deckbar, Dorothee; Löbrich, Markus; Yang, Qin; Khanna, Kum Kum; Worman, Howard J.; Pandita, Tej K.

doi:10.1128/mcb.01676-12

Cited by 104 publications

(102 citation statements)

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“…Other studies have also implicated LA/C in the regulation of DNA synthesis. Recently, LA and LC were shown to play a significant role in the restart of replication forks following replicative stress and the recruitment of DNA repair complexes, but a direct interaction between LA/C and newly replicating chromatin was not demonstrated (5). We found that LA/C interacted directly with newly replicated DNA at multiple origins of replication, in contrast to LB1, which interacted only weakly with a minority of origins.…”

Section: Discussionmentioning

confidence: 41%

“…2A), suggesting that most origins of replication are affected by the reduction of LB1 levels. Several studies have suggested that lamins can interact with sites of replication to affect the efficiency of replication; however, a direct biochemical interaction of lamins with sites of replication has not been demonstrated (5,23,33). In order to determine if the LB1-dependent decrease in DNA replication was due to a direct interaction of lamins with newly replicating DNA, we performed the BrdU ChIP procedure with nonsilenced cells followed by immunoblotting of the precipitated proteins for the presence of replication factors and lamins.…”

Section: ϫ3mentioning

confidence: 99%

“…The altered lamins produced because of these mutations have been shown to affect interactions with lamin-binding proteins, cause telomere dysfunction, disrupt the epigenetic regulation and organization of chromatin, and alter gene expression (4,5). Accumulation of the unprocessed form of LA, called pre-LA, is also linked to the activation of DNA repair-regulating factors and checkpoint kinases, which possibly contribute to impaired cell cycle progression and replication arrest (6,7).…”

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Role of Lamin B1 in Chromatin Instability

Butin‐Israeli

Adam

Jain

et al. 2015

Molecular and Cellular Biology

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c Nuclear lamins play important roles in the organization and structure of the nucleus; however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. The S phase delay appears to be due to the stalling and collapse of replication forks. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions. The lamins are type V intermediate-filament proteins found primarily within the nuclei of metazoan cells. The lamins play important roles in providing mechanical support and shape to the nucleus and also participate in various chromatin-associated processes, including DNA replication, polymerase (Pol) II transcription, DNA repair, mitotic-spindle formation, response to oxidative stress, and chromosome positioning (1). However, the exact mechanisms by which lamins are involved in these pathways remain largely unclear. Vertebrate cells express two types of lamins, the A types, lamins A and C (LA and LC), and the B types, lamin B1 (LB1) and lamin B2 (LB2). LA and LC are expressed in developmentally regulated patterns from a single gene, LMNA, by alternative splicing. In contrast, LB1 and LB2 are expressed from two different genes, with at least one B-type lamin being expressed in all cell types throughout development and differentiation (2).A-type lamins have received the most attention in recent years because of the hundreds of mutations identified in LMNA that cause a spectrum of rare diseases known as laminopathies (3). The altered lamins produced because of these mutations have been shown to affect interactions with lamin-binding proteins, cause telomere dysfunction, disrupt the epigenetic regulation and organization of chromatin, and alter gene expression (4, 5). Accumulation of the unprocessed form of LA, called pre-LA, is also linked to the activation of DNA repair-regulating factors and checkpoint kinases, which possibly contribute to impaired cell cycle progression and replication arrest (6, 7). Pre-LA has also been reported to cause the accumulation of unrepaired DNA because of delayed recruitment of DNA repair proteins to DNA damage sites (8). In contrast to the num...

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Section: Discussionmentioning

confidence: 41%

Section: ϫ3mentioning

confidence: 99%

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confidence: 99%

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Role of Lamin B1 in Chromatin Instability

Butin‐Israeli

Adam

Jain

et al. 2015

Molecular and Cellular Biology

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“…A nucleoplasmic population of lamin A was reported to colocalize within chromatin foci that contain the replication proteins PCNA and DNA polymerase ␦; these foci are believed to represent sites of DNA replication in early S phase (5,6). Interestingly, lamin A/C deficiency enhances replication stress in cells treated with interstrand cross-linking agents, suggesting a role for lamin A/C in the recovery of stalled replication forks (53). Unprocessed prelamin A likely interferes with DNA replication by sequestering replication and repair factors (9,39,54).…”

Section: Discussionmentioning

confidence: 99%

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome

Wheaton

Campuzano

et al. 2017

Molecular and Cellular Biology

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Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to ␥H2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.KEYWORDS HGPS, progerin, senescence, aging, p53, telomere T he study of children with rare accelerated aging (progeria) syndromes has provided insight into the normal process of aging. Hutchinson-Gilford progeria syndrome (HGPS) is caused by a heterozygous, autosomal dominant mutation in LMNA (1), the gene that encodes lamin A, a key structural protein in the nuclear lamina. The lamina, a protein network that lines the inner nuclear membrane, provides structural support for the nucleus and is important for chromatin attachment, DNA replication, nuclear organization, and gene transcription in ways that are not completely understood. Farnesylation of prelamin A at the C terminus allows targeting to the inner nuclear membrane, where it is subsequently cleaved by the zinc metalloproteinase Zmpste24 to release mature lamin A into the lamina and nucleoplasm. In HGPS, the most common LMNA mutation (G608G) activates a cryptic splice donor site in exon 11, resulting in prelamin A mRNA with a 150-bp internal deletion, leading to a 50-amino-acid truncation in a region that contains the Zmpste24 cleavage site (reviewed in reference 2). The mutant lamin, termed progerin, retains the farnesyl lipid anchor, interferes with the integrity of the nuclear lamina, and causes the formation of misshapen nuclei. The retention of progerin on the inner nuclear membrane interferes with the function and normal distribution of lamin A, causing a loss of peripheral heterochromatin, increased DNA damage, impaired recruitment of DNA repair proteins to sites of DNA damage (3), and persistent activation of DNA damage response proteins (4). In addition to its

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“…For example, one of down-regulated protein, programmed cell death protein 6 (PDCD6), also called apoptosislinked gene 2 protein (ALG2), functions as calciumbinding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death (Suzuki et al 2012;Yoon et al 2012). Besides their multifunction, four proteins, ATP-dependent RNA helicase DDX39A, complement component 1 Q subcomponent-binding protein (SF2p32), Lamin-A/C, and far upstream element-binding Protein 2 are involved in pre-mRNA splicing or as splicing factors to play an important role in DNA damage and repair (Dittmer and Misteli 2011;Filippov et al 2007;Marengo and Wassarman 2008;Singh et al 2013). Replication factor C (RFC) subunit 5 is down regulated, suggesting that RFC and Pol d may be synergistically involved in DNA repair events on lagging or leading strand with their altered functions by the down-regulation of their smallest subunits.…”

Section: Discussionmentioning

confidence: 99%

Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells

et al. 2014

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Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol d) results in an inter-conversion between heterotetramer (Pol d4) and heterotrimer (Pol d3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNAdamaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736 ± 13 proteins in non-treated control group and 741 ± 19 protein spots in HUtreated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

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Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

Cited by 104 publications

References 50 publications

Role of Lamin B1 in Chromatin Instability

Role of Lamin B1 in Chromatin Instability

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome

Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells

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