Initiation and Continuation of DNA Replication are not Associated with the Nuclear Envelope in Mammalian Cells

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

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Overexpression of kin17 protein disrupts nuclear morphology and inhibits the growth of mammalian cells

Journal of Cell Science ◽

10.1242/jcs.112.19.3215 ◽

1999 ◽

Vol 112 (19) ◽

pp. 3215-3224 ◽

Cited By ~ 1

Author(s):

P. Kannouche ◽

J.F. Angulo

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Reca Protein ◽

S Phase ◽

Expression Vectors ◽

Nuclear Morphology ◽

Functional Domain ◽

Genetic Response ◽

Cycle Progression

UVC or ionizing radiation of mammalian cells elicits a complex genetic response that allows recovery and cell survival. Kin17 gene, which is highly conserved among mammals, is upregulated during this response. Kin17 gene encodes a 45 kDa protein which binds to DNA and presents a limited similarity with a functional domain of the bacterial RecA protein. Kin17 protein is accumulated in the nucleus of proliferating fibroblasts and forms intranuclear foci. Using expression vectors, we show that overexpression of kin17 protein inhibits cell-cycle progression into S phase. Our results indicate that growth inhibition correlates with disruption of the nuclear morphology which seems to modify the intranuclear network required during the early steps of DNA replication. We report that a mutant encoding a protein deleted from the central domain of kin17 protein enhanced these effects whereas the deletion of the C-terminal domain considerably reduced them. These mutants will be used to elucidate the molecular mechanism by which kin17 protein alters cell growth and DNA replication.

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Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.400-406.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 400-406

Author(s):

W P Deng ◽

J A Nickoloff

Keyword(s):

Dna Replication ◽

Mismatch Repair ◽

Mammalian Cells ◽

Strand Break ◽

Wild Type ◽

Heteroduplex Dna ◽

Frameshift Mutations ◽

Single Strand Annealing ◽

Extrachromosomal Recombination ◽

Strand Annealing

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

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The Protective Role of Dormant Origins in Response to Replicative Stress

International Journal of Molecular Sciences ◽

10.3390/ijms19113569 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3569 ◽

Cited By ~ 11

Author(s):

Lilas Courtot ◽

Jean-Sébastien Hoffmann ◽

Valérie Bergoglio

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Genome Stability ◽

Replication Timing ◽

Protective Role ◽

Entire Genome ◽

Replicative Stress ◽

A Cell ◽

The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

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