Nuclear organization of DNA replication in primary mammalian cells

Kennedy, Brian K.; Barbie, David A.; Classon, Marie; Dyson, Nicholas J.; Harlow, Ed

doi:10.1101/gad.842600

Cited by 272 publications

(257 citation statements)

References 53 publications

(74 reference statements)

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“…Based on these data, we suggest that telomerase extends the life span of HGPS cells through its reparative function, regulation of proliferation, and its role in chromatin maintenance. 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).…”

Section: Discussionmentioning

confidence: 99%

“…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 structural role, the colocalization of nucleoplasmic lamin A with the replication proteins PCNA and DNA polymerase ␦ within nuclear foci suggests an involvement of lamin A in DNA replication (5,6). Additionally, the interaction of lamin A with LAP2A␣ (7) and RB (8) is important for regulation of cell cycle entry.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

mentioning

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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“…This directly links pRb, DNA repair and UV radiationinduced apoptosis. pRb family members have been shown to co-localize with chromatin assembly factor-1 (CAF-1) (Kennedy et al, 2000), a protein important in NER of UV-induced lesions (Game and Kaufman, 1999;Martini et al, 1998). Indeed, in yeast, CAF-1 mutants are hypersensitive to UV radiation (Game and Kaufman, 1999).…”

Section: Discussionmentioning

confidence: 99%

“…The importance of the co-localization is still unknown. In addition, pRb has been shown to localize to sites of DNA synthesis in early S phase (Kennedy et al, 2000), although traditionally, pRb is known to be a transcriptional repressor. These novel associations and binding partners for pRb point to a possible role for pRb in DNA synthesis and repair, and would constitute entirely new pathways for pRb outside of its well known role as a cell cycle regulator.…”

Section: Discussionmentioning

confidence: 99%

Lack of functional pRb results in attenuated recovery of mRNA synthesis and increased apoptosis following UV radiation in human breast cancer cells

et al. 2002

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Lack of functional pRb results in attenuated recovery of mRNA synthesis and increased apoptosis following UV radiation in human breast cancer cells. We have previously demonstrated that a human breast cancer cell line, MDA-MB-468, which lacks the retinoblastoma protein (pRb), is particularly sensitive to low doses of ultraviolet (UV) radiation. These cells are 15 ± 20-fold more sensitive to UV radiation than cells with wild-type pRb. In order to understand the mechanisms of the high apoptotic response of MDA-MB-468 cells to UV radiation, we examined the e ects of UV on these cells with regards to both membrane-mediated events and DNA damage. We found that MDA-MB-468 cells were resistant to all ligandinduced death receptor signaling. In addition, although UV activated caspase 8 in MDA-MB-468 cells, a peptide inhibitor of caspase 8 failed to inhibit UV-induced apoptosis. We then tested the possibility that nuclear events mediated the enhanced sensitivity to UV-induced apoptosis in these cells. Unlike UV-resistant cells, MDA-MB-468 cells were unable to recover mRNA synthesis after 5 J/m 2 UVC. We also found that the pRb-null DU-145 cells similarly had attenuated recovery of mRNA synthesis after UV radiation. In UV-resistant cells with wild-type pRb, the inactivation of pRb with HPV-16 E7 resulted in signi®cant inhibition in their ability to recover mRNA synthesis and increased levels of apoptosis following UV radiation. Furthermore, pRb-null cells were de®cient in repair of UV radiation-induced DNA damage. These data suggest that the sensitivity of MDA-MB-468 cells to UV radiation is due to defects in repair of DNA damage and recovery of mRNA synthesis rather than to membrane death receptor pathways. Inactivation of pRb may contribute to an increased sensitivity to UV radiation by attenuating repair of DNA lesions and recovery of mRNA synthesis following UV radiation.

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“…A-type lamins localize to peripheral regions underlying the nuclear envelope and also concentrate at perinucleolar foci (7,31). They are hypothesized to affect gene expression at both peripheral and perinucleolar sites due to interactions with transcription factors involved in differentiation, such as the retinoblastoma protein (pRB) (26,41,42,44,51,59,67).…”

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confidence: 99%

Stabilization of the Retinoblastoma Protein by A-Type Nuclear Lamins Is Required for INK4A-Mediated Cell Cycle Arrest

Nitta¹,

Jameson²,

Kudlow

et al. 2006

Molecular and Cellular Biology

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Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16 ink4a , we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16 ink4a -mediated G 1 arrest. Reintroduction of lamin A, lamin C, or pRB restores p16 ink4a -responsiveness to Lmna ؊/؊ cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna ؊/؊ cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16 ink4a responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.A-type lamins are intermediate filament proteins that have been linked to the organization and maintenance of nuclear structure. In most differentiated tissues, A-type lamins (predominantly lamins A and C), along with lamin B, comprise the meshwork underlying the inner nuclear membrane known as the nuclear lamina (63). Unlike lamin C, lamin A contains a carboxy-terminal CaaX motif and must undergo a series of posttranslational modifications to form the mature lamin A (77). In particular, the cysteine of the CaaX motif in lamin A is isoprenylated, followed by cleavage of the aaX and carboxymethylation of the C-terminal cysteine (72). Lastly, a second cleavage event by Zmpste24 removes the last 15 amino acids to yield the mature lamin A (3, 13, 74).Mutations within the LMNA gene cause a variety of human disorders collectively known as laminopathies. These include progeria syndromes and dystrophies associated with skeletal muscle and adipose tissue (5,10,15,19,45,58). Mice lacking A-type lamins or deficient in processing lamin A develop skeletal and cardiac muscular dystrophies, thus confirming the importance of A-type lamins for muscle differentiation and/or maintenance (52, 65). The mechanism by which mutations in A-type lamins generate tissue-specific disorders is unknown. However, one model posits that lamin A/C regulates gene expression in differentiated tissue by coordinating the activity of key transcription factors.A-type lamins localize to p...

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Nuclear organization of DNA replication in primary mammalian cells

Cited by 272 publications

References 53 publications

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

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

Lack of functional pRb results in attenuated recovery of mRNA synthesis and increased apoptosis following UV radiation in human breast cancer cells

Stabilization of the Retinoblastoma Protein by A-Type Nuclear Lamins Is Required for INK4A-Mediated Cell Cycle Arrest

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