Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis

Spann, Timothy P.; Moir, Robert D.; Goldman, Anne E.; Stick, Reimer; Goldman, Robert D.

doi:10.1083/jcb.136.6.1201

Cited by 258 publications

(263 citation statements)

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“…Previous studies demonstrated that disruption of lamin organization by a dominant-negative lamin A mutant (ΔNLA), missing the first 33 amino acids of human lamin A, repressed DNA replication. This was associated with the redistribution of DNA replication proteins RFC and PCNA away from chromatin to form nuclear aggregates along with the nuclear lamins (37). In addition, accumulation of prelamin A has been shown to interfere with PCNA function and cause replication stress (9).…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…A-type lamins have also been found in association with transcription factors (Dreuillet et al, 2002;Lloyd et al, 2002). Furthermore, disruption of normal lamin organization by microinjection of a dominant-negative lamin A mutant results in inhibition of both DNA replication (Spann et al, 1997) and RNA polymerase II-dependent transcription (Spann et al, 2002) suggesting a role for lamin A in both these processes. Finally, lamins have the ability to bind directly to DNA (Stierle et al, 2003) and to chromatin (Glass et al, 1993;Taniura et al, 1995) and indirectly via associations with proteins containing a LEM box (Lee et al, 2001;Martins et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Aging of Hutchinson–Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis

Bridger

Kill

2004

Experimental Gerontology

157

137

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Hutchinson -Gilford progeria syndrome is a rare genetic disorder that mimics certain aspects of aging prematurely. Recent work has revealed that mutations in the lamin A gene are a cause of the disease. We show here that cellular aging of Hutchinson -Gilford progeria syndrome fibroblasts is characterised by a period of hyperproliferation and terminates with a large increase in the rate of apoptosis. The occurrence of cells with abnormal nuclear morphology reported by others is shown to be a result of cell division since the fraction of these abnormalities increases with cellular age. Similarly, the proportion of cells with an abnormal or absent A-type lamina increases with age. These data provide clues as to the cellular basis for premature aging in HGPS and support the view that cellular senescence and tissue homeostasis are important factors in the normal aging process. q 2004 Elsevier Inc. All rights reserved.

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“…The proteins expressed by using this vector contained an N-terminal His 6 tag followed by 6 kDa of laminin rod (38). The lamin fragments were expressed in NovaBlue DE3 bacteria cotransfected with pLys-S (Novagen) and induced with 1 mM isopropyl ␤-D-thiogalactoside for 2-4 h. The protein fragments were solubilized from inclusion bodies in column buffer (6 M urea͞10 mM Tris⅐HCl, pH 8.0͞10 mM DTT͞2 mM EDTA), purified on a Mono Q column by FPLC (Amersham Pharmacia), and eluted in column buffer containing 0-1.0 M NaCl (11). Column fractions containing the lamin fragments were identified by SDS͞PAGE and either dialyzed into PB or further purified by using nitrilotriacetic acid-agarose (Qiagen) and then dialyzed into PB.…”

Section: Bacterial Expression and Purification Of Wild-type And Mutanmentioning

confidence: 99%

“…Nuclei were suspended in NWB, fixed with either 2% ethylene glycol bis(succinimidylsuccinate) (Pierce) or 2.0% formaldehyde for 10 min, and spun onto poly(L-lysine)-coated coverslips through 20% sucrose in NWB, as described in ref. 11. The coverslips were washed with PBS containing 0.1% Nonidet P-40, washed again with PBS, and overlayed with primary Abs diluted 1:200 (11).…”

Section: Bacterial Expression and Purification Of Wild-type And Mutanmentioning

confidence: 99%

Functions and dysfunctions of the nuclear lamin Ig-fold domain in nuclear assembly, growth, and Emery–Dreifuss muscular dystrophy

Shumaker

Lopez‐Soler²,

Adam

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The non-␣-helical C terminus of Xenopus lamin B3 (LB3T) inhibits the polymerization of lamin B3 in vitro and prevents the assembly of nuclei in Xenopus egg interphase extracts. To more precisely define the functions of LB3T in nuclear assembly, we have expressed subdomains of LB3T and determined their effects on nuclear assembly in Xenopus extracts. The results demonstrate that the Ig-fold motif (LB3T-Ig) is sufficient to inhibit lamin polymerization in vitro. Addition of the LB3T-Ig to egg extracts before the introduction of chromatin prevents chromatin decondensation and the assembly of the lamina, membranes, and pore complexes comprising the nuclear envelope. When added to assembled nuclei, LB3T-Ig prevents the further incorporation of lamin B3 into the endogenous lamina and blocks nuclear growth. The introduction of a point mutation in LB3T-Ig (R454W; LB3T-IgRW), known to cause Emery-Dreifuss muscular dystrophy when present in lamin A, does not inhibit lamin polymerization, chromatin decondensation, or nuclear assembly and growth. These results shed light on the specific alterations in lamin functions attributable to a known muscular dystrophy mutation and provide an experimental framework for revealing the effects of other mutations causing a wide range of laminopathies. ''tail'' domains (3, 4). The crystal structures of three small regions within these domains are known. These structures include coils 1A and 2B of the central rod (5) and the Ig-like fold (Ig-fold) present in the tail (6, 7). The ␣-helical rod domain is required for the formation of coiled-coil lamin dimers, which are the building blocks of higher order lamin structures. The function(s) of the Ig-fold in the nuclear lamins is unknown. In other systems, however, this motif is involved in protein-protein, protein-DNA, and protein-phospholipid interactions (6).Within nuclei, lamins are found throughout the nucleoplasm and are concentrated in the lamina, which forms an interface between the inner nuclear membrane and chromatin (8,9). During the cell cycle, the organization of lamins in interphase nuclei is not static. For example, during S-phase, lamins associate with replication factors such as proliferating cell nuclear antigen and replication factor C (10-12), whereas in mitosis, lamins are phosphorylated by the mitotic kinase cdk1 (13), causing their disassembly during nuclear envelope breakdown (14 -16). Lamins are subsequently dephosphorylated as they repolymerize around chromatin during nuclear assembly in daughter cells (17,18). The process of lamin polymerization is initiated in the anaphase-telophase transition during which B-type lamins begin to assemble on chromosomes and continues into early G 1 (9).Insights into lamin functions have been obtained from studies of the cell-free assembly of nuclei in Xenopus egg interphase extracts (19). For example, addition of the non-␣-helical Cterminal domain of Xenopus lamin B3 (LB3T) inhibits lamin polymerization, chromatin decondensation, and nuclear membrane and pore assembly (20). Other lam...

show abstract

Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis

Cited by 258 publications

References 60 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

Aging of Hutchinson–Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis

Functions and dysfunctions of the nuclear lamin Ig-fold domain in nuclear assembly, growth, and Emery–Dreifuss muscular dystrophy

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