Genetic mouse models to investigate cell cycle regulation

Early studies on cell cycle regulation were based on experiments in model systems (Yeast, Xenopus, Starfish, Drosophila) and have shaped the way we understand many events that control the cell cycle. Although these model systems are of great value, the last decade was highlighted by studies done in human cells and using in vivo mouse models. Mouse models are irreplaceable tools for understanding the genetics, development, and survival strategies of mammals. New developments in generating targeting vectors and mutant mice have improved our approaches to study cell cycle regulation and cancer. Here we summarize the most recent advances of mouse model approaches in dissecting the mechanisms of cell cycle regulation and the relevance to human disease. W. Li and S. Kotoshiba contributed equally.     

Characterization of two cellobiose dehydrogenases and comparison of their contributions to total activity in Neurospora crassa

Cellobiose dehydrogenase production by Neurospora crassa was investigated in this study. N. crassa has two putative cellobiose dehydrogenase (CDH) genes (cdh) in its genome. CDH was produced only under cellulolytic conditions. Deletion of nc-cdh1 eliminated almost all of the strain’s CDH activity, whereas the deletion of nc-cdh2 had little effect on total extracellular CDH activity, which indicates that NC-CDH1 is a major contributor to overall CDH activity. The homologous expression of nc-cdh1 and nc-cdh2 under the control of the constitutive D-glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter enabled recombinant CDH production under non-cellulolytic conditions. Both NC-CDH1 and NC-CDH2 produced by N. crassa were successfully purified and characterized for the first time. NC-CDH1 and NC-CDH2 have molecular weights of 100 kDa and 130 kDa, respectively. When their N-linked glycans were removed by N-glycosidase F treatment, both enzymes showed a molecular weight of 95 kDa. Although NC-CDH2 lacks the cellulose-binding module and contributed marginally to total CDH activity in N. crassa, NC-CDH2 has specific activity similar to that of NC-CDH1 (7.93 vs. 8.89 IU mg⁻¹), and it has a much lower K_m value than that of NC-CDH1 (5.79 vs. 25.72 μM). The lower activity contribution of NC-CDH2 in the wild-type strain may results from its lower enzyme production.     

Homologous Recombination is Activated at Early Time Points Following Exposure to Cobalt Chloride Induced Hypoxic Conditions in Saccharomyces cerevisiae

DNA repair functions are essential for the maintenance of genetic integrity and are regulated in response to both environmental and chemical stressors in mammalian and yeast cells in culture. The inhibitory effect of limited O₂ availability on DNA repair functions in general and on homologous recombination (HR) in particular, correlates with increased chromosomal abnormalities in hypoxic cancer cells. Given the above, we have investigated the effects of CoCl₂,––a hypoxia mimetic agent on HR and genetic aberrations in Saccharomyces cerevisiae. Our studies demonstrate that both acute and chronic exposure to CoCl₂ activated HR and increased genetic aberrations in S. cerevisiae D7 cells. At early time points following addition of CoCl₂ to the growth media, cells were briefly arrested in the G1-S boundary concomitant with a transient increase in Rad52-GFP foci formation and induction of low levels of DNA damage. The mode of action of CoCl₂ is thus similar to that of DNA synthesis inhibitors, the later are known to induce HR and cause G1-S arrest. We propose that the activation of HR in the presence of the hypoxia mimetic agent may be attributed to the replication stress and/or DNA damage induced by the stressor.     

Structural analysis of RecA protein-DNA complexes by fluorescence-detected linear dichroism: absence of structural change of filament for pairing of complementary DNA strands

We have developed a simple measuring system for fluorescence-detected linear dichroism and applied it to the structural analysis of the RecA-DNA complex filaments, which are intermediates of the homologous recombination reaction. Taking advantage of the selectivity of fluorescence signals, we distinguished the linear dichroism signals of ethidium bromide and tryptophan residues in the RecA-DNA-ethidium bromide complex, whereas the conventional (absorption-detected) linear dichroism measurement provides only the sum of the signals because signals overlap each other and that of DNA. We further observed that the tryptophan residue at position 290 of RecA in the RecA-DNA-adenosine-5'-O-(3-thiotriphosphate) complex was oriented parallel to the long axis of the filament, in good agreement with the previous site-specific linear dichroism analysis, and that this orientation was not significantly modified by the pairing of the complementary DNA strand. These results suggest that the pairing reaction occurs without a large structural change of the RecA filament.     

Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein

The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.     

Poetry in motion: Increased chromosomal mobility after DNA damage

Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae, examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution.     

Leishmania major: Disruption of signal peptidase type I and its consequences on survival, growth and infectivity

Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival.The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.     

基因置换发展研究综述

基因置换,又称基因替代作用,它指的是遗传信息单向的从一个序列向其同源序列传递的过程。近年来,人们对遗传重组的发生机制、功能及影响有了新的认识,尤其是发现"重组"造成的基因置换远超过以前认识。与遗传重组不同,基因置换有可能改变等位基因频率,搅乱等位基因的组合,降低连锁不平衡的程度,影响核苷酸多样性的即时效应和长期的效应。这些发现有助于去理解基因置换的机制,去探索基因置换对物种进化及遗传多样性更深远的影响,如基因置换对染色体基因结构和功能有哪些影响及基因置换对更深层次的生物进化起什么样的作用等。本文对基因置换进行了综合论述,重点介绍了基因置换的当前发展趋势,并以基因四联子为模型,总结了基因置换事件的推断,并对基因置换的研究前景进行了讨论。     

Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells

Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination.     

黑曲霉pepB基因缺失菌株的构建及其功能分析

以黑曲霉(Aspergillus niger)GICC2773基因组DNA为模板,用PCR方法分别扩增pepB基因中的上游约1．4kb和下游约1．3kb两段DNA序列,将此两段序列按同一方向分别插入质粒pMW1中潮霉素抗性基因(hph)表达单元的5′和3′端,构建成重组质粒pMW1-pepB,用于通过同源重组靶向破坏基因组中的pepB基因。同源重组则采用原生质体-PEG方法,将酶切pMW1-pepB得到的线性片段转化A．niger GICC2773菌株,通过潮霉素选择平板得到62个Hgy抗性转化子,然后采用PCR方法从这些抗性转化子中筛选到1个由于同源重组产生的pepB基因缺失突变菌株pepB29。功能分析显示该突变株的酸性蛋白酶活性有明显下降,外源蛋白漆酶的分泌表达有所提高。