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ABSTRACT: PDSS2 is a gene that encodes one of the two subunits of trans-prenyl diphosphate synthase that is essential for ubiquinone biosynthesis. It is known that mutations in PDSS2 can cause primary ubiquinone deficiency in humans and a similar disease in mice. Cerebellum is the most often affected organ in ubiquinone deficiency, and cerebellar atrophy has been diagnosed in many infants with this disease. In this study, two Pdss2 conditional knockout mouse lines directed by Pax2-cre and Pcp2-cre were generated to investigate the effect of ubiquinone deficiency on cerebellum during embryonic development and in adulthood, respectively. The Pdss2(f/-); Pax2-cre mouse recapitulates some symptoms of ubiquinone deficiency in infants, including severe cerebellum hypoplasia and lipid accumulation in skeletal muscles at birth. During early cerebellum development (E12.5-14.5), Pdss2 knockout initially causes the delay of radial glial cell growth and neuron progenitor migration, so the growth of mutant cerebellum is retarded. During later development (E15.5-P0), increased ectopic apoptosis of neuroblasts and impaired cell proliferation result in the progression of cerebellum hypoplasia in the mutant. Thus, the mutant cerebellum contains fewer neurons at birth, and the cells are disorganized. The developmental defect of mutant cerebellum does not result from reduced Fgf8 expression before E12.5. Electron microscopy reveals mitochondrial defects and increased autophagic-like vacuolization that may arise in response to abnormal mitochondria in the mutant cerebellum. Nevertheless, the mutant mice die soon after birth probably due to cleft palate and micrognathia, which may result from Pdss2 knockout caused by ectopic Pax2-cre expression in the first branchial arch. On the other hand, the Pdss2(f/-); Pcp2-cre mouse is healthy at birth but gradually loses cerebellar Purkinje cells and develops ataxia-like symptoms at 9.5 months; thus this conditional knockout mouse may serve as a model for ubiquinone deficiency in adult patients. In conclusion, this study provides two mouse models of Pdss2 based ubiquinone deficiency. During cerebellum development, Pdss2 knockout results in severe cerebellum hypoplasia by impairing cell migration and eliciting ectopic apoptosis, whereas Pdss2 knockout in Purkinje cells at postnatal stages leads to the development of cerebellar ataxia.
Neurobiology of Disease 08/2011; 45(1):219-33. · 5.40 Impact Factor
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ABSTRACT: Protein ubiquitination is a crucial component of the DNA damage response. To study the mechanism of the DNA damage-induced ubiquitination pathway, we analyzed the impact of the loss of two E3 ubiquitin ligases, RNF8 and Chfr. Notably, DNA damage-induced activation of ATM kinase is suppressed in cells deficient in both RNF8 and Chfr (double-knockout, or DKO), and DKO mice develop thymic lymphomas that are nearly diploid but harbor clonal chromosome translocations. Moreover, DKO mice and cells are hypersensitive to ionizing radiation. We present evidence that RNF8 and Chfr synergistically regulate histone ubiquitination to control histone H4 Lys16 acetylation through MRG15-dependent acetyltransferase complexes. Through these complexes, RNF8 and Chfr affect chromatin relaxation and modulate ATM activation and DNA damage response pathways. Collectively, our findings demonstrate that two chromatin-remodeling factors, RNF8 and Chfr, function together to activate ATM and maintain genomic stability in vivo.
Nature Structural & Molecular Biology 06/2011; 18(7):761-8. · 12.71 Impact Factor
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ABSTRACT: During spermatogenesis, global nucleosome removal occurs where histones are initially replaced by transition proteins and subsequently by protamines. This chromatin reorganization is thought to facilitate the compaction of the paternal genome into the sperm head and to protect the DNA from damaging agents. Histone ubiquitination has been suggested to be important for sex chromosome inactivation during meiotic prophase and nucleosome removal at postmeiotic stages. However, the mechanisms regulating these ubiquitin-mediated processes are unknown. In this study, we investigate the role of the ubiquitin ligase RNF8 during spermatogenesis and find that RNF8-deficient mice are proficient in meiotic sex chromosome inactivation (MSCI) but deficient in global nucleosome removal. Moreover, we show that RNF8-dependent histone ubiquitination induces H4K16 acetylation, which may be an initial step in nucleosome removal. Thus, our results show that RNF8 plays an important role during spermatogenesis through histone ubiquitination, resulting in trans-histone acetylation and global nucleosome removal.
Developmental cell 03/2010; 18(3):371-84. · 13.36 Impact Factor
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ABSTRACT: BRCA1 is a well-established tumor suppressor gene, which is frequently mutated in familial breast and ovarian cancers. The gene product of BRCA1 functions in a number of cellular pathways that maintain genomic stability, including DNA damage-induced cell cycle checkpoint activation, DNA damage repair, protein ubiquitination, chromatin remodeling, as well as transcriptional regulation and apoptosis. In this review, we discuss recent advances regarding our understanding of the role of BRCA1 in tumor suppression and DNA damage response, including DNA damage-induced cell cycle checkpoint activation and DNA damage repair.
Protein & Cell 02/2010; 1(2):117-23.
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ABSTRACT: Targeted gene repair directed by single-stranded oligodeoxynucleotides (ssODNs) offers a promising tool for biotechnology and gene therapy. However, the methodology is currently limited by its low frequency of repair events, variability, and low viability of "corrected" cells. In this study, we showed that during ssODN-mediated gene repair reaction, a significant population of corrected cells failed to divide, and were much more prone to undergo apoptosis, as marked by processing of caspases and PARP-1. In addition, we found that apoptotic cell death triggered by ssODN-mediated gene repair was largely independent of the ATM/ATR kinase. Furthermore, we examined the potential involvement of the mismatch repair (MMR) proteins in this "correction reaction-induced" cell death. Result showed that while defective MMR greatly enhanced the efficiency of gene correction, compromising the MMR system did not yield any viable corrected clone, indicating that the MMR machinery, although plays a critical role in determining ssODN-directed repair, was not involved in the observed cellular genotoxic responses.
Oligonucleotides 09/2009; 19(3):281-6. · 2.80 Impact Factor
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ABSTRACT: Polo-like kinase 1 (Plk1) belongs to a family of conserved serine/threonine kinases with a polo-box domain, which have similar but non-overlapping functions in the cell cycle progression. Plk1 plays a key role to ensure the normal mitosis. Interestingly, overexpression of Plk1 is associated with tumor development and could serve as a prognostic marker for many cancers. Due to Plk1 overexpression, several Plk1 inhibitors have been developed and tested for the cancer treatment. However, in a recent study, it has been suggested that down-regulation of Plk1 could also induce aneuploidy and tumor formation in vivo. Therefore, a normal level of Plk1 is important for mitosis. And caution should be taken when Plk1 inhibitors are used in the clinical trial and their side effects including tumorigenesis should be carefully evaluated.
Cell Division 02/2009; 4:4. · 3.00 Impact Factor
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ABSTRACT: Histone ubiquitination participates in multiple cellular processes, including the DNA damage response. However, the molecular mechanisms involved are not clear. Here, we have identified that RAP80/UIMC1 (ubiquitin interaction motif containing 1), a functional partner of BRCA1, recognizes ubiquitinated histones H2A and H2B. The interaction between RAP80 and ubiquitinated histones H2A and H2B is increased following DNA damage. Since RAP80 facilitates BRCA1's translocation to DNA damage sites, our results indicate that ubiquitinated histones H2A and H2B could be upstream partners of the BRCA1/RAP80 complex in the DNA damage response. Moreover, we have found that RNF8 (ring finger protein 8), an E3 ubiquitin ligase, regulates ubiquitination of both histones H2A and H2B. In RNF8-deficient mouse embryo fibroblasts, ubiquitination of both histones H2A and H2B is dramatically reduced, which abolishes the DNA damage-induced BRCA1 and RAP80 accumulation at damage lesions on the chromatin. Taken together, our results suggest that ubiquitinated histones H2A and H2B may recruit the BRCA1 complex to DNA damage lesions on the chromatin.
Molecular and cellular biology 12/2008; 29(3):849-60. · 6.06 Impact Factor
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ABSTRACT: Aurora A is a serine/threonine kinase that functions in various stages of mitosis. Accumulating evidence has demonstrated that gene amplification and overexpression of Aurora A are linked to tumorigenesis, suggesting that Aurora A is an oncogene. In addition, Aurora A overexpression has been used as a negative prognostic marker, because it is associated with resistance to anti-mitotic agents commonly used for cancer therapy. To understand the physiological functions of Aurora A, we generated Aurora A knock-out mice. Aurora A null mice die early during embryonic development before the 16-cell stage. These Aurora A null embryos have defects in mitosis, particularly in spindle assembly, supporting critical functions of Aurora A during mitotic transitions. Interestingly, Aurora A heterozygosity results in a significantly increased tumor incidence in mice, suggesting that Aurora A may also act as a haploinsufficient tumor suppressor. Consistently, Aurora A heterozygous mouse embryonic fibroblasts have higher rates of aneuploidy. We further discovered that VX-680, an Aurora kinase inhibitor currently in phase II clinical trials for cancer treatment, could induce aneuploidy in wild type mouse embryonic fibroblasts. We conclude that a balanced Aurora A level is critical for maintaining genomic stability and one needs to be fully aware of the potential side effects of anti-cancer therapy based on the use of Aurora A-specific inhibitors.
Journal of Biological Chemistry 10/2008; 283(46):31785-90. · 4.77 Impact Factor
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ABSTRACT: Polo-like kinases (Plks) are serine/threonine kinases that are highly conserved in organisms from yeasts to humans. Previous reports have shown that Plk1 is critical for all stages of mitosis and may play a role in DNA replication during S phase. While much work has focused on Plk1, little is known about the physiological function of Plk1 in vivo. To address this question, we generated Plk1 knockout mice. Plk1 homozygous null mice were embryonic lethal, and early Plk1(-/-) embryos failed to survive after the eight-cell stage. Immunocytochemistry studies revealed that Plk1-null embryos were arrested outside the mitotic phase, suggesting that Plk1 is important for proper cell cycle progression. It has been postulated that Plk1 is a potential oncogene, due to its overexpression in a variety of tumors and tumor cell lines. While the Plk1 heterozygotes were healthy at birth, the incidence of tumors in these animals was threefold greater than that in their wild-type counterparts, demonstrating that the loss of one Plk1 allele accelerates tumor formation. Collectively, our data support that Plk1 is important for early embryonic development and may function as a haploinsufficient tumor suppressor.
Molecular and cellular biology 10/2008; 28(22):6870-6. · 6.06 Impact Factor
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ABSTRACT: The lamda phage Red recombination system has been used to modify plasmid, bacterial artificial chromosome (BAC), and chromosomal DNA in a highly precise and versatile manner Linear double-stranded DNA fragments or synthetic single-stranded oligonucleotides (SSOs) with short flanking homologies (<50 bp) to the target loci can be used as substrates to direct changes, including point mutations, insertions, and deletions. In attempts to explore mechanistic bases under this recombination process, we and others have previously identified factors that influence SSO-mediated single base substitutions. In this report, we focus our study on SSO-mediated deletion on plasmids. We found that SSOs as short as 63 bp were sufficient to mediate deletion as long as 2 kb with efficiency higher than 1%. Strand bias was consistently observed, and SSOs with sequences identical to the nascent lagging strand during replication always resulted in higher efficiency. Unlike SSO-mediated single nucleotide substitution, homology on each side of SSO flanking the fragment to be deleted was important for successful deletion, and abolishing the host methyl-directed mismatch repair (MMR) system did not lead to detectable changes in deletion efficiency. Finally, we showed that by optimizing its design, SSO-mediated deletion was efficient enough to make it possible to manipulate plasmids without selectable markers.
BioTechniques 03/2008; 44(2):217-20, 222, 224. · 2.67 Impact Factor
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ABSTRACT: The lambda-Red-mediated recombination has been exploited as an efficient means for DNA manipulation. We previously reported that replication plays a pivotal role during this process. Replication direction dictates strand bias, such that single stranded oligonucleotide (SSO) with sequence corresponding to the nascent lagging strand directs higher levels of recombinant formation compared to its complementary SSO. In addition, the Escherichia coli methyl-directed mismatch repair system impedes efficient SSO-mediated site-specific gene repair. However, the role of transcription in determining strand bias and recombination efficiency is unclear. To address the potential role of transcriptional processes, we constructed plasmid substrates that harbor a mutant antibiotic reporter under the control of an inducible promoter. We found that transcription activation can promote recombinant formation to more than 10-folds whilst it has negligible effect on strand bias. Our findings provide evidence for a role of transcription in SSO-mediated gene repair process.
Biochemical and Biophysical Research Communications 03/2007; 353(1):33-9. · 2.48 Impact Factor
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Nan Yang,
Julian A Tanner,
Bo-Jian Zheng,
Rory M Watt,
Ming-Liang He, Lin-Yu Lu,
Jie-Qing Jiang,
Ka-To Shum,
Yong-Ping Lin,
Kin-Ling Wong,
Marie C M Lin,
Hsiang-Fu Kung,
Hongzhe Sun,
Jian-Dong Huang
Angewandte Chemie International Edition 02/2007; 46(34):6464-8. · 13.45 Impact Factor
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ABSTRACT: Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the 'annealing-integration' model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a lambda-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.
Nucleic Acids Research 02/2006; 34(21):6183-94. · 8.03 Impact Factor
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Julian A Tanner,
Bo-Jian Zheng,
Jie Zhou,
Rory M Watt,
Jie-Qing Jiang,
Kin-Ling Wong,
Yong-Ping Lin, Lin-Yu Lu,
Ming-Liang He,
Hsiang-Fu Kung,
Andreas J Kesel,
Jian-Dong Huang
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ABSTRACT: Bananins are a class of antiviral compounds with a unique structural signature incorporating a trioxa-adamantane moiety covalently bound to a pyridoxal derivative. Six members of this class of compounds: bananin, iodobananin, vanillinbananin, ansabananin, eubananin, and adeninobananin were synthesized and tested as inhibitors of the SARS Coronavirus (SCV) helicase. Bananin, iodobananin, vanillinbananin, and eubananin were effective inhibitors of the ATPase activity of the SCV helicase with IC50 values in the range 0.5-3 microM. A similar trend, though at slightly higher inhibitor concentrations, was observed for inhibition of the helicase activities, using a FRET-based fluorescent assay. In a cell culture system of SCV, bananin exhibited an EC50 of less than 10 microM and a CC50 of over 300 microM. Kinetics of inhibition are consistent with bananin inhibiting an intracellular process or processes involved in SCV replication.
Chemistry & Biology 04/2005; 12(3):303-11. · 5.83 Impact Factor
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ABSTRACT: Recombinogenic engineering methodology, also known as recombineering, utilizes homologous recombination to create targeted changes in cellular DNA with great specificity and flexibility. In Escherichia coli, the Red recombination system from bacteriophage lambda has been used successfully to modify both plasmid and chromosomal DNA in a highly efficient manner, using either a linear double-stranded DNA fragment or a synthetic single-stranded oligonucleotide (SSO). The current model for Red/SSO-mediated recombination involves the SSO first annealing to a transient, single-stranded region of DNA before being incorporated into the chromosome or plasmid target. It has been observed previously, in both eukaryotes and prokaryotes, that mutations in the two strands of the DNA double helix are 'corrected' by complementary SSOs with differing efficiencies. Here we investigate further the factors that influence the strand bias as well as the overall efficiency of Red/SSO-mediated recombination in E.coli. We show that the direction of DNA replication and the nature of the SSO-encoded mismatch are the main factors dictating the recombinational strand bias. However, the influence that the SSO-encoded mismatch exerts upon the recombinational strand bias is abolished in E.coli strains that are defective in mismatch repair (MMR). This reflects the fact that different base-base mispairs are corrected by the mutS/H/L-dependent MMR pathway with differing efficiencies. Furthermore, our data indicate that transcription has negligible influence on the strand bias. These results demonstrate for the first time that the interplay between DNA replication and MMR has a major effect on the efficiency and strand bias of Red/SSO-mediated recombination in E.coli.
Nucleic Acids Research 12/2003; 31(22):6674-87. · 8.03 Impact Factor
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ABSTRACT: The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.
Journal of Biological Chemistry 11/2003; 278(41):39578-82. · 4.77 Impact Factor
