Maturation‐associated Dbf4 expression is essential for mouse zygotic DNA replication

Cdc7 is an S‐phase‐promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one‐cell embryos at zygotic S‐phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one‐cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi‐mediated knockdown of maternal Dbf4 expression prevents the maturation‐associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one‐cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3′ untranslated region (3′UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.     

两种冷冻保护剂对小鼠2-细胞胚冷冻效果的比较

乙二醇（ETG）和1,2-丙二醇（PROH）具有高细胞渗透性和低毒性特点,常被用于人及多种哺乳动物早期胚胎冷冻保存。为了比较ETG和PROH对小鼠2-细胞胚的冷冻保护效果,本试验分别采用这两种冷冻保护剂,对小鼠2-细胞胚进行冷冻保存,并采用冻后体外培养和囊胚移植进行冷冻效果检测。结果表明,PROH组胚胎解冻后胚胎存活率与ETG组无显著差异,但PROH组4-细胞胚发育率和囊胚发育率显著高于ETG组（82．7％vs．64．6％,61．2％vs．29．1％,P〈0．01）。囊胚移植结果表明,2-细胞胚胎冻存后能够发育为正常的后代,PROH组和ETG组的囊胚移植后妊娠产仔率无统计学差异（P〉0．05）,但均显著低于对照组（P〈0．05）。为了分析两组胚胎冻存后损伤情况,埘解冻后的胚胎细胞微丝进行检测,结果显示ETG组微丝受损的胚胎数高于PROH组。本研究结果证明采用PROH作为冷冻保护剂冷冻保存小鼠2-细胞胚的冻存效果优于ETG[动物学报54（6）：1098—1105,2008]。     

Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

Histone deacetylases 1 and 2 regulate DNA replication and DNA repair: potential targets for genome stability-mechanism-based therapeutics for a subset of cancers

Histone deacetylases 1 and 2 (HDAC1,2) belong to the class I HDAC family, which are targeted by the FDA-approved small molecule HDAC inhibitors currently used in cancer therapy. HDAC1,2 are recruited to DNA break sites during DNA repair and to chromatin around forks during DNA replication. Cancer cells use DNA repair and DNA replication as survival mechanisms and to evade chemotherapy-induced cytotoxicity. Hence, it is vital to understand how HDAC1,2 function during the genome maintenance processes (DNA replication and DNA repair) in order to gain insights into the mode-of-action of HDAC inhibitors in cancer therapeutics. The first-in-class HDAC1,2-selective inhibitors and Hdac1,2 conditional knockout systems greatly facilitated dissecting the precise mechanisms by which HDAC1,2 control genome stability in normal and cancer cells. In this perspective, I summarize the findings on the mechanistic functions of class I HDACs, specifically, HDAC1,2 in genome maintenance, unanswered questions for future investigations and views on how this knowledge could be harnessed for better-targeted cancer therapeutics for a subset of cancers.     

Identification of novel initiation sites for human DNA replication around ARSH1, a previously characterized yeast replicator

Replication of mammalian chromosomes depends on the activation of a large number of origins of DNA replication distributed along the chromosomes. We have focused our attention on a human DNA region, named ARSH1, localized to chromosome 2, that had been previously shown to act as an episomal origin in the yeast Saccharomyces cerevisiae. In the present study we have used a nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 5 kb region encompassing ARSH1. This analysis applied to a 1-1.4 kb nascent DNA strand fraction isolated from normal skin fibroblasts revealed the presence of two major initiations sites surrounding the ARSH1 region. With an equivalent DNA fraction obtained from HeLa cells, in addition to these sites, a broad initiation profile was observed which included the ARSH1 region. This DNA region however was not sufficient to support episomal replication of an ARSH1-containing plasmid transfected into HeLa cells.     

HMGB1 protein inhibits DNA replication in vitro: a role of the acetylation and the acidic tail

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.     

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis

Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G₁ transition and for pre-RC maintenance in G₁ phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.     

Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

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The essential requirement for Runx1 in the development of the sternum

Runx1 is highly expressed in chondroprogenitor and osteoprogenitor cells and in vitro experiments suggest that Runx1 is important in the early stages of osteoblast and chondrocyte differentiation. However, because Runx1 knockout mice are early embryonic lethal due to failure of hematopoiesis, the role of Runx1 in skeletogenesis remains unclear. We studied the role of Runx1 in skeletal development using a Runx1 reversible knockout mouse model. By crossing with Tie2-Cre deletor mice, Runx1 expression was selectively rescued in the endothelial and hematopoietic systems but not in the skeleton. Although Runx1^Re/Re embryos survived until birth and had a generally normal skeleton, the development of mineralization in the sternum and some skull elements was significantly disrupted. In contrast to wild-type embryos, the sternum of E17.5 Runx1^Re/Re embryos showed high levels of Sox-9 and collagen type II expression and lack of development of hypertrophic chondrocytes. In situ hybridization analysis demonstrated that, in contrast to the vertebrae and long bones, the sternum of wild-type embryos expresses high levels of Runx1, but not Runx2, the master regulator of skeletogenesis. Thus, although Runx1 is not essential for major skeletal development, it does play an essential role in the development of the sternum and some skull elements.