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Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

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Reduced chromatin bound Mcm2 in the flo intestine.(A) Western analysis showing reduced levels of chromatin bound Mcm2 in 75 hpf and 96 hpf flo intestine compared with wild type siblings. By contrast, levels of chromatin bound Mcm3 (B) and Mcm4 (C) in the flo intestine are comparable to wild type. Multiple bands (*) corresponding to phospho-Mcm3 and phospho-Mcm4 are recognized by the antibodies directed against the native proteins in wt and flo samples.
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pgen-1000240-g007: Reduced chromatin bound Mcm2 in the flo intestine.(A) Western analysis showing reduced levels of chromatin bound Mcm2 in 75 hpf and 96 hpf flo intestine compared with wild type siblings. By contrast, levels of chromatin bound Mcm3 (B) and Mcm4 (C) in the flo intestine are comparable to wild type. Multiple bands (*) corresponding to phospho-Mcm3 and phospho-Mcm4 are recognized by the antibodies directed against the native proteins in wt and flo samples.

Mentions: Recently, the Mcm2-7 complex, a central component of the DNA replication helicase, was shown to bind Elys and promote its loading onto chromatin at the M-G1 transition of the cell cycle [22]. Reconstitution of the NPC complex was postulated to promote the nuclear import of Geminin, a Cdt1 inhibitor, at the onset of S-phase, thus preventing reloading of Mcm2-7 onto chromatin and DNA re-replication. These data were the first to link replication licensing with NPC assembly. Despite the absence of nuclear pores in flo retinal and intestinal epithelial cells, we found no evidence of promiscuous firing of licensed replication origins in these highly replicative cells, as arrest in G1 rather than S phase was evident in both tissues (Table S2). However, biochemical analyses did reveal an effect of Elys deficiency on Mcm-chromatin interactions. These showed significantly reduced levels of chromatin bound Mcm2 were present in the flo intestine at 75 hpf and 96 hpf (Figure 7A and Figure S6). By contrast, levels of Mcm3 and Mcm4 in flo mutants were either normal or minimally reduced at both time points (Figure 7B–7C), and Mcm2 levels were only minimally reduced in intestinal cells of slim jim mutants, which have reduced proliferation and undergo cell death (Figure S6). Levels of phospho-Mcm4, which are elevated in response to replication stress [45],[46] were also normal in cells derived from irradiated flo intestines (Figure S6). Together, these data link Elys deficiency with reduced chromatin loading or maintenance of chromatin binding of selected Mcm2-7 complex proteins.


Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

Reduced chromatin bound Mcm2 in the flo intestine.(A) Western analysis showing reduced levels of chromatin bound Mcm2 in 75 hpf and 96 hpf flo intestine compared with wild type siblings. By contrast, levels of chromatin bound Mcm3 (B) and Mcm4 (C) in the flo intestine are comparable to wild type. Multiple bands (*) corresponding to phospho-Mcm3 and phospho-Mcm4 are recognized by the antibodies directed against the native proteins in wt and flo samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570612&req=5

pgen-1000240-g007: Reduced chromatin bound Mcm2 in the flo intestine.(A) Western analysis showing reduced levels of chromatin bound Mcm2 in 75 hpf and 96 hpf flo intestine compared with wild type siblings. By contrast, levels of chromatin bound Mcm3 (B) and Mcm4 (C) in the flo intestine are comparable to wild type. Multiple bands (*) corresponding to phospho-Mcm3 and phospho-Mcm4 are recognized by the antibodies directed against the native proteins in wt and flo samples.
Mentions: Recently, the Mcm2-7 complex, a central component of the DNA replication helicase, was shown to bind Elys and promote its loading onto chromatin at the M-G1 transition of the cell cycle [22]. Reconstitution of the NPC complex was postulated to promote the nuclear import of Geminin, a Cdt1 inhibitor, at the onset of S-phase, thus preventing reloading of Mcm2-7 onto chromatin and DNA re-replication. These data were the first to link replication licensing with NPC assembly. Despite the absence of nuclear pores in flo retinal and intestinal epithelial cells, we found no evidence of promiscuous firing of licensed replication origins in these highly replicative cells, as arrest in G1 rather than S phase was evident in both tissues (Table S2). However, biochemical analyses did reveal an effect of Elys deficiency on Mcm-chromatin interactions. These showed significantly reduced levels of chromatin bound Mcm2 were present in the flo intestine at 75 hpf and 96 hpf (Figure 7A and Figure S6). By contrast, levels of Mcm3 and Mcm4 in flo mutants were either normal or minimally reduced at both time points (Figure 7B–7C), and Mcm2 levels were only minimally reduced in intestinal cells of slim jim mutants, which have reduced proliferation and undergo cell death (Figure S6). Levels of phospho-Mcm4, which are elevated in response to replication stress [45],[46] were also normal in cells derived from irradiated flo intestines (Figure S6). Together, these data link Elys deficiency with reduced chromatin loading or maintenance of chromatin binding of selected Mcm2-7 complex proteins.

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Show MeSH
Related in: MedlinePlus