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Spartan deficiency causes genomic instability and progeroid phenotypes.

Maskey RS, Kim MS, Baker DJ, Childs B, Malureanu LA, Jeganathan KB, Machida Y, van Deursen JM, Machida YJ - Nat Commun (2014)

Bottom Line: However, the physiological relevance of Spartan has not been established.Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death.These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA.

ABSTRACT
Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

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Effects of Sprtn KO on DNA replication forks.(a) Schematic representation of DNA fiber assays. MEFs were treated with MeOH or 4-OHT for 48 h and sequentially labelled with IdU and CldU to mark ongoing replication. A picture of a representative replication track is shown. At least 100 fibres were scored for each sample in all of the DNA fiber experiments. (b) A box plot showing distribution of the lengths of CldU tracts in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. NS, not significant (P=0.829, two-tailed unpaired t-test). (c) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with SprtnF/− MEFs with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. (d–f) Effect of ultraviolet irradiation on replication forks. Experiments were performed as in c using SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant (d), wild-type human Spartan, the PIP* or the UBZ* mutant (e), and wild-type mouse Spartan or the SHP* mutant (f). Horizontal red lines in c–f indicate median values.
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f5: Effects of Sprtn KO on DNA replication forks.(a) Schematic representation of DNA fiber assays. MEFs were treated with MeOH or 4-OHT for 48 h and sequentially labelled with IdU and CldU to mark ongoing replication. A picture of a representative replication track is shown. At least 100 fibres were scored for each sample in all of the DNA fiber experiments. (b) A box plot showing distribution of the lengths of CldU tracts in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. NS, not significant (P=0.829, two-tailed unpaired t-test). (c) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with SprtnF/− MEFs with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. (d–f) Effect of ultraviolet irradiation on replication forks. Experiments were performed as in c using SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant (d), wild-type human Spartan, the PIP* or the UBZ* mutant (e), and wild-type mouse Spartan or the SHP* mutant (f). Horizontal red lines in c–f indicate median values.

Mentions: To examine the effect of Sprtn inactivation on DNA replication more directly, we measured movement of replication forks in Sprtn−/− MEFs using DNA fiber assays, in which replication tracts were sequentially labelled with the thymidine analogues IdU and CldU (Fig. 5a). Lengths of CldU-labelled DNA in Sprtn−/− MEFs were not significantly different from those of Sprtn+/+ MEFs (Fig. 5b), suggesting that the majority of replication forks are not affected by Spartan loss, an observation consistent with the normal S-phase progression in Sprtn−/− cells (Supplementary Fig. 1b).


Spartan deficiency causes genomic instability and progeroid phenotypes.

Maskey RS, Kim MS, Baker DJ, Childs B, Malureanu LA, Jeganathan KB, Machida Y, van Deursen JM, Machida YJ - Nat Commun (2014)

Effects of Sprtn KO on DNA replication forks.(a) Schematic representation of DNA fiber assays. MEFs were treated with MeOH or 4-OHT for 48 h and sequentially labelled with IdU and CldU to mark ongoing replication. A picture of a representative replication track is shown. At least 100 fibres were scored for each sample in all of the DNA fiber experiments. (b) A box plot showing distribution of the lengths of CldU tracts in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. NS, not significant (P=0.829, two-tailed unpaired t-test). (c) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with SprtnF/− MEFs with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. (d–f) Effect of ultraviolet irradiation on replication forks. Experiments were performed as in c using SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant (d), wild-type human Spartan, the PIP* or the UBZ* mutant (e), and wild-type mouse Spartan or the SHP* mutant (f). Horizontal red lines in c–f indicate median values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269170&req=5

f5: Effects of Sprtn KO on DNA replication forks.(a) Schematic representation of DNA fiber assays. MEFs were treated with MeOH or 4-OHT for 48 h and sequentially labelled with IdU and CldU to mark ongoing replication. A picture of a representative replication track is shown. At least 100 fibres were scored for each sample in all of the DNA fiber experiments. (b) A box plot showing distribution of the lengths of CldU tracts in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. NS, not significant (P=0.829, two-tailed unpaired t-test). (c) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with SprtnF/− MEFs with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. (d–f) Effect of ultraviolet irradiation on replication forks. Experiments were performed as in c using SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant (d), wild-type human Spartan, the PIP* or the UBZ* mutant (e), and wild-type mouse Spartan or the SHP* mutant (f). Horizontal red lines in c–f indicate median values.
Mentions: To examine the effect of Sprtn inactivation on DNA replication more directly, we measured movement of replication forks in Sprtn−/− MEFs using DNA fiber assays, in which replication tracts were sequentially labelled with the thymidine analogues IdU and CldU (Fig. 5a). Lengths of CldU-labelled DNA in Sprtn−/− MEFs were not significantly different from those of Sprtn+/+ MEFs (Fig. 5b), suggesting that the majority of replication forks are not affected by Spartan loss, an observation consistent with the normal S-phase progression in Sprtn−/− cells (Supplementary Fig. 1b).

Bottom Line: However, the physiological relevance of Spartan has not been established.Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death.These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA.

ABSTRACT
Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

Show MeSH
Related in: MedlinePlus