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. 2009;4(1):e4184.
doi: 10.1371/journal.pone.0004184. Epub 2009 Jan 15.

DNA polymerase delta is required for early mammalian embryogenesis (VSports注册入口)

Arikuni Uchimura  1 , Yuko Hidaka, Takahiro Hirabayashi, Masumi Hirabayashi, Takeshi Yagi
Affiliations

"VSports" DNA polymerase delta is required for early mammalian embryogenesis

Arikuni Uchimura et al. PLoS One. 2009.

Abstract (V体育ios版)

Background: In eukaryotic cells, DNA polymerase delta (Poldelta), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination VSports手机版. However, the physiological role of the Poldelta in mammalian development has not been thoroughly investigated. .

Methodology/principal findings: To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Poldelta-null (Pold1(-/-)) mice and D400A exchanged Poldelta (Pold1(exo/exo)) mice. The D400A exchange caused deficient 3'-5' exonuclease activity in the Poldelta protein. In Poldelta-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1(-/-) mice suffered from peri-implantation lethality. Although Pold1(-/-) blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Poldelta participates in DNA replication during mouse embryogenesis V体育安卓版. In Pold1(exo/exo) mice, although heterozygous Pold1(exo/+) mice were normal and healthy, Pold1(exo/exo) and Pold1(exo/-) mice suffered from tumorigenesis. .

Conclusions: These results clearly demonstrate that DNA polymerase delta is essential for mammalian early embryogenesis and that the 3'-5' exonuclease activity of DNA polymerase delta is dispensable for normal development but necessary to suppress tumorigenesis. V体育ios版.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of two kinds of Pold1 mutated mice by gene targeting strategy.
(A) Schematic of the Pold1 domain structure and locations of the modifications with a Pold1 gene targeting method. (B) Structure of the targeting vector and partial restriction map of the murine Pold1 locus before and after homologous recombination and Cre mediated recombination. Exons are represented by vertical black boxes and introns by intervening horizontal lines. * indicate D400A substitution. The genomic fragment used as a probe for Southern blotting is indicated by the horizontal blue bar. Restriction enzymes: D indicates DraI; N indicates NheI. PCR primers P0 and P2 were used to screen for homologous recombinant ES cells. PCR primers P1, P2 and P3 were used for genotyping. (C) PCR screening for homologous recombinants. A 2430-bp band was amplified with the primer pairs P0–P2, specific to the homologous recombinant. Lane 1–3 are independent homologous recombinant ES clones; lane 4 is a wild-type ES clone. (D) PCR genotyping of mice tail DNA. A 499-bp wild-type band and a 654-bp Pold1 exo band were amplified with primer pairs P1–P3 and a 334-bp Pold1 − band was amplified with primer pairs P1–P2. These primers are shown in (B). (E) Southern blot analysis to identify each Pold1 allele with mouse tail DNA. In DraI digestion, the 15.9-kb fragment corresponds to the wild-type allele, the 16.0-kb fragment corresponds to the Pold1 exo allele (the two overlap) and the 6.4-kb fragment corresponds to the Pold1 − allele. In NheI digestion, the 14.6-kb fragment represents the wild-type allele while the 6.0-kb fragment represents both Pold1 − and Pold1 exo alleles. (F) DNA sequencing of RT-PCR products from heterozygous Pold1 +/− and Pold1 exo/+ ES cells. Aspartic acid, D, was substituted with alanine, A, in Pold1 exo alleles. (G) Schematic represents the results of nucleotide sequencing the 3′RACE products derived from Pold1 − alleles in Pold1 +/− ES cells. Primers P4 and P5 were used for 3′RACE repeated amplification. 3′RACE products from Pold1 − alleles were distinguished by means of sequencing the D400A substitution. (H) Immunoblot analysis of two pairs of extracts derived from E12.5 whole embryos performed with anti-Pold1 and anti-β-actin (control) antibodies; one pair compares Pold1 +/− and Pold1 +/+ from the same litter of embryos while the other compares Pold1 exo/exo and Pold1 +/+ embryos.
Figure 2
Figure 2. Morphological analysis of blastocysts and outgrowths from blastocysts.
(A) The appearance of mutant embryos. E3.5 and E4.5 embryos were flushed out from the uteri. E4.5 embryos were cultured for 2 hr in M2 medium. After taking photographs under bright-field conditions, E3.5 and E4.5 embryos were genotyped by PCR. (B) Impaired proliferation of Pold1-deficient blastocyst outgrowths in vitro. Blastocysts were harvested at E3.5, cultured in vitro for several days, and subsequently genotyped by PCR. Black horizontal bar indicates 50 µm.
Figure 3
Figure 3. Pold1 deficient embryos showed defective DNA synthesis.
DNA synthesis was observed by determining BrdU incorporation in cultured blastocysts in vitro. (A) E3.5 embryos were cultured for 24 hr, then cultured in the presence of BrdU for 3 hr. After indirect immunofluorescence using anti-BrdU (green) and anti-pHH3 (red) antibodies and counterstained with DAPI (blue), embryos were genotyped by PCR. (B) E3.5 embryos were cultured for 3 days, cultured in the presence of BrdU for 3 hr, and processed for immunostaining against BrdU (green) and pHH3 (red) and DAPI counterstaining (blue). The genotype of each embryo was subsequently determined by PCR. White horizontal bar indicates 50 µm.
Figure 4
Figure 4. Spontaneous apoptosis detected in Pold1 deficient embryos.
Spontaneous apoptosis was analyzed by TUNEL assay. TUNEL-positive cells appeared in Pold1-deficient blastocyst outgrowths. After taking a photo, the genotype of each embryo was determined by PCR. White horizontal bar indicates 50 µm.
Figure 5
Figure 5. Lack of Polδ 3′–5′ exonuclease activity causes tumors.
(A) Swollen thymus in 4-month old Pold1 exo/− mice. Th: thymus, H: heart. (B) Tail with nodule in 14-month old Pold1 exo/− mice.
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