Lagging-Strand, Early-Labelling, and Two-Dimensional Gel Assays Suggest Multiple Potential Initiation Sites in the Chinese Hamster Dihydrofolate Reductase Origin

There is general agreement that DNA synthesis in the single-copy and amplified dihydrofolate reductase (DHFR) loci of CHO cells initiates somewhere within the 55-kb spacer region between the DHFR and 2BE2121 genes. However, results of lagging-strand, early-labelling fragment hybridization (ELFH), and PCR-based nascent-strand abundance assays have been interpreted to suggest a very narrow zone of initiation centered at a single locus known as ori-β, while two-dimensional (2-D) gel analyses suggest that initiation can occur at any of a large number of potential sites scattered throughout the intergenic region. The results of a leading-strand assay and two intrinsic labelling techniques are compatible with a broad initiation zone in which ori-β and a second locus (ori-γ) are somewhat preferred. To determine how these differing views are shaped by differences in experimental manipulations unrelated to the biology itself, we have applied the lagging-strand, ELFH, neutral-neutral, and/or neutral-alkaline 2-D gel assays to CHOC 400 cell populations synchronized and manipulated in the same way. In our experiments, the lagging-strand assay failed to identify a template strand switch at ori-β; rather, we observed a gradual, undulating change in hybridization bias throughout the intergenic spacer, with hybridization to the two templates being approximately equal near a centered matrix attachment region. In the ELFH assay, all of the fragments in the 55-kb intergenic region were labelled in the first few minutes of the S phase, with the regions encompassing ori-β and ori-γ being somewhat preferred. Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multiple locations in the intergenic spacer. Thus, the results of all existing replicon-mapping methods that have been applied to the amplified DHFR locus in CHOC 400 cells are consistent with a model in which two somewhat preferred subzones reside in a larger zone of multiple potential initiation sites in the intergenic region.     

二氢叶酸还原酶基因在斑马鱼神经系统发育过程中的作用

目的观察二氢叶酸还原酶基因(DHFR)功能阻抑斑马鱼胚胎的颅脑部发育情况,初步探讨二氢叶酸还原酶基因在斑马鱼神经系统发育过程中的作用。方法采用显微注射吗啡啉修饰的反义核苷酸(MO)的方法进行DHFR表达阻抑。胚胎发育至受精后48hpf观察胚胎的颅部发育情况,在60hpf时经石蜡切片进一步观察胚胎的脑发育状况。利用胚胎整体原位杂交的方法检测影响神经系统发育的关键因子ngn1和huc的表达情况。结果显微注射MO可成功的进行DHFR表达阻抑。DHFR表达阻抑组胚胎存在颅脑部发育明显异常和ngn1、huc的表达强度明显减弱,且与显微注射的MO剂量呈正相关。结论DHFR在斑马鱼颅脑发育中有重要作用;其功能阻抑可导致胚胎颅脑部发育异常,其机理与ngn1和huc的的表达减弱有关。     

AAA+ ATPases in the Initiation of DNA Replication

All cellular organisms and many viruses rely on large, multi-subunit molecular machines, termed replisomes, to ensure that genetic material is accurately duplicated for transmission from one generation to the next. Replisome assembly is facilitated by dedicated initiator proteins, which serve to both recognize replication origins and recruit requisite replisomal components to the DNA in a cell-cycle coordinated manner. Exactly how imitators accomplish this task, and the extent to which initiator mechanisms are conserved among different organisms have remained outstanding issues. Recent structural and biochemical findings have revealed that all cellular initiators, as well as the initiators of certain classes of double-stranded DNA viruses, possess a common adenine nucleotide-binding fold belonging to the ATPases Associated with various cellular Activities (AAA+) family. This review focuses on how the AAA+ domain has been recruited and adapted to control the initiation of DNA replication, and how the use of this ATPase module underlies a common set of initiator assembly states and functions. How biochemical and structural properties correlate with initiator activity, and how species-specific modifications give rise to unique initiator functions, are also discussed.     

Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.     

Initiation of DNA Replication in Cells of Higher Eukaryotes

In this review, the problems concerning initiation of DNA replication in higher eukaryotes are discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Initiation of Chromosomal DNA Replication in Mammalian Cell-Free Systems

Chromosomal DNA replication is a fundamental part of the cell division cycle of eukaryotes, and its disruption often leads to genome instability and cancer. A focus for regulation is the initiation of the first replication forks, marking the transition from G1 to S phase. Direct biochemical investigation of the establishment and further progression of chromosomal DNA replication in human somatic cell nuclei has become possible through a cell-free system that obeys cell cycle control. Since its development less than a decade ago, several modifications and adaptations of the original system have been reported, which have led to temporal resolution of replication complex assembly and to the identification of novel DNA replication factors. Here, I will review the different systems, highlight fundamental differences and unifying concepts, and discuss their potential for understanding chromosomal DNA replication in somatic mammalian cells.     

Replication of the Bacterial Chromosome: Location of New Initiation Sites After Irradiation

New loci of replication along the bacterial chromosome are observed after irradiation of Escherichia coli. It was conjectured that, after X-irradiation, the new initiation site was random with respect to the fixed-origin, whereas, after ultraviolet light exposure, it was selective and appeared to be from the fixed-origin. Evidence presented here shows that, after X-irradiation of E. coli, the new initiation site(s) for the onset of deoxyribonucleic acid replication is induced at chromosomal regions not restricted to the fixed-origin. After ultraviolet light exposure, the new initiation site is preferentially from the fixed-origin. In these studies amino acid starvation was used to synchronize chromosome replication and to allow for differential radioisotopic labeling of the chromosomal origin and terminus. To facilitate interpretation, growing cells actively replicating their chromosome were compared with cells lacking growth points at the time of irradiation. The role of these new replication sites in the observed kinetics of deoxyribonucleic acid replication following X-ray or ultraviolet light exposure is discussed.     

真核细胞染色体DNA复制起始及复制叉稳定性维持的机制

在真核生物中,DNA复制在染色体上特定的多位点起始．当细胞处在晚M及G1期,多个复制起始蛋白依次结合到DNA复制源,组装形成复制前复合体．pre．RC在Gl-S的转折期得到激活,随后,多个直接参与DNA复制又形成的蛋白结合到DNA复制源,启动DNA的复制,形成两个双向的DNA复制又．在染色体上,移动的DNA复制又经常会碰到复制障碍（二级DNA结构、一些蛋白的结合位点、损伤的碱基等）而暂停下来,此时,需要细胞周期检验点的调控来稳定复制叉,否则,会导致复制又垮塌及基因组不稳定．本文就真核细胞染色体DNA复制起始的机制,以及复制又稳定性的维持机制进行简要综述．     

Differentiation of the DnaA-oriC Subcomplex for DNA Unwinding in a Replication Initiation Complex

In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.     

Mapping of Replication Origins in the X Inactivation Center of Vole Microtus levis Reveals Extended Replication Initiation Zone

DNA replication initiates at specific positions termed replication origins. Genome-wide studies of human replication origins have shown that origins are organized into replication initiation zones. However, only few replication initiation zones have been described so far. Moreover, few origins were mapped in other mammalian species besides human and mouse. Here we analyzed pattern of short nascent strands in the X inactivation center (XIC) of vole Microtus levis in fibroblasts, trophoblast stem cells, and extraembryonic endoderm stem cells and confirmed origins locations by ChIP approach. We found that replication could be initiated in a significant part of XIC. We also analyzed state of XIC chromatin in these cell types. We compared origin localization in the mouse and vole XIC. Interestingly, origins associated with gene promoters are conserved in these species. The data obtained allow us to suggest that the X inactivation center of M. levis is one extended replication initiation zone.     

Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

The hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) is a model for examining how protein dynamics contribute to enzymatic function. The relationship between functional motions and enzyme evolution has attracted significant attention. Recent studies on N23PP Escherichia coli DHFR (ecDHFR) mutant, designed to resemble parts of the human enzyme, indicated a reduced single turnover rate. NMR relaxation dispersion experiments with that enzyme showed rigidification of millisecond Met-20 loop motions (Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., Benkovic, S. J., and Wright, P. E. (2011) Science 332, 234–238). A more recent study of this mutant, however, indicated that fast motions along the reaction coordinate are actually more dispersed than for wild-type ecDHFR (WT). Furthermore, a double mutant (N23PP/G51PEKN) that better mimics the human enzyme seems to restore both the single turnover rates and narrow distribution of fast dynamics (Liu, C. T., Hanoian, P., French, T. H., Hammes-Schiffer, S., and Benkovic, S. J. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 10159–11064). Here, we measured intrinsic kinetic isotope effects for both N23PP and N23PP/G51PEKN double mutant DHFRs over a temperature range. The findings indicate that although the C-H→C transfer and dynamics along the reaction coordinate are impaired in the altered N23PP mutant, both seem to be restored in the N23PP/G51PEKN double mutant. This indicates that the evolution of G51PEKN, although remote from the Met-20 loop, alleviated the loop rigidification that would have been caused by N23PP, enabling WT-like H-tunneling. The correlation between the calculated dynamics, the nature of C-H→C transfer, and a phylogenetic analysis of DHFR sequences are consistent with evolutionary preservation of the protein dynamics to enable H-tunneling from well reorganized active sites.     

Dual Sites of Protein Initiation Control the Localization and Myristoylation of Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system, which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. In mammals, one gene encodes two forms of the reductase, one targeted to the cytosol and the other to mitochondria. The cytosolic form displays faster mobility than the mitochondrial form, suggesting a lower molecular weight for the former. The apparent size difference and targeting to two cellular compartments had been proposed to result from differential splicing of mRNA. We now show that differential targeting is effected by use of two initiation sites, one of which includes a mitochondrial targeting sequence, whereas the other does not. We also demonstrate that the mass of the cytosolic form is not less than that of the mitochondrial form; the faster mobility of cytosolic form is due to its myristoylation. Lipidation of methionine sulfoxide reductase A occurs in the mouse, in transfected tissue culture cells, and even in a cell-free protein synthesis system. The physiologic role of myristoylation of MsrA remains to be elucidated.     

Multiple and Specific Initiation of T4 DNA Replication

Partially replicated T4 DNA molecules (PRM) whose parental or progeny DNA was labeled with bromodeoxyuridine BUdR was analyzed by gradual shearing followed by CsCl banding of the sheared product. Analysis of PRM containing 18-mum replicated DNA showed that each replicated region was 3- to 6-mum long, indicating three to 6 replicative sites per molecule. Analysis of PRM containing 9-mum replicated DNA similarly indicated two to three replicated regions per molecule. DNA from the replicated regions of PRM containing 10-mum replicated DNA ("donor") was hybridized to DNA from mature phage ("recipient"), and the resulting hybrid was subjected to digestion with exonuclease I. The extent of protection of the recipient and more efficient self-annealing of progeny fragments from PRM indicated that the replicated regions represented 8 to 10 nonrandom locations of the genome. Possible significance of multiple sites for initiation of DNA replication is discussed.     

质粒的复制特性和整合后在发动大肠杆菌染色体复制中对recA基因的依赖性的研究

大肠杆菌的温度敏感的DNA复制发动突变型dnaA46在42℃中不能生长而形成菌落,质粒或噬菌体的整合使它能生长。前文[2]报道了F质粒整合后所发动的染色体复制依赖于recA基因。本文报道5种质粒和2种噬菌体整合后所发动的染色体复制对recA基因的依赖性的研究结果。实验结果说明,依赖与否和它们在游离状态中复制方向无关,从而否定了recA基因的作用是把质粒或噬菌体的游离状态的单向复制发动机构改变为整合状态的双向复制发动饥构这一假设。     

Beyond DnaA: The Role of DNA Topology and DNA Methylation in Bacterial Replication Initiation

The replication of chromosomal DNA is a fundamental event in the life cycle of every cell. The first step of replication, initiation, is controlled by multiple factors to ensure only one round of replication per cell cycle. The process of initiation has been described most thoroughly for bacteria, especially Escherichia coli, and involves many regulatory proteins that vary considerably between different species. These proteins control the activity of the two key players of initiation in bacteria: the initiator protein DnaA and the origin of chromosome replication (oriC). Factors involved in the control of the availability, activity, or oligomerization of DnaA during initiation are generally regarded as the most important and thus have been thoroughly characterized. Other aspects of the initiation process, such as origin accessibility and susceptibility to unwinding, have been less explored. However, recent findings indicate that these factors have a significant role. This review focuses on DNA topology, conformation, and methylation as important factors that regulate the initiation process in bacteria. We present a comprehensive summary of the factors involved in the modulation of DNA topology, both locally at oriC and more globally at the level of the entire chromosome. We show clearly that the conformation of oriC dynamically changes, and control of this conformation constitutes another, important factor in the regulation of bacterial replication initiation. Furthermore, the process of initiation appears to be associated with the dynamics of the entire chromosome and this association is an important but largely unexplored phenomenon.     

Early Events in the Pathogenesis of Foot-and-Mouth Disease in Pigs; Identification of Oropharyngeal Tonsils as Sites of Primary and Sustained Viral Replication

A time-course study was performed to elucidate the early events of foot-and-mouth disease virus (FMDV) infection in pigs subsequent to simulated natural, intra-oropharyngeal, inoculation. The earliest detectable event was primary infection in the lingual and paraepiglottic tonsils at 6 hours post inoculation (hpi) characterized by regional localization of viral RNA, viral antigen, and infectious virus. At this time FMDV antigen was localized in cytokeratin-positive epithelial cells and CD172a-expressing leukocytes of the crypt epithelium of the paraepiglottic tonsils. De novo replication of FMDV was first detected in oropharyngeal swab samples at 12 hpi and viremia occurred at 18–24 hpi, approximately 24 hours prior to the appearance of vesicular lesions. From 12 through 78 hpi, microscopic detection of FMDV was consistently localized to cytokeratin-positive cells within morphologically characteristic segments of oropharyngeal tonsil crypt epithelium. During this period, leukocyte populations expressing CD172a, SLA-DQ class II and/or CD8 were found in close proximity to infected epithelial cells, but with little or no co-localization with viral proteins. Similarly, M-cells expressing cytokeratin-18 did not co-localize with FMDV proteins. Intra-epithelial micro-vesicles composed of acantholytic epithelial cells expressing large amounts of structural and non-structural FMDV proteins were present within crypts of the tonsil of the soft palate during peak clinical infection. These findings inculpate the paraepiglottic tonsils as the primary site of FMDV infection in pigs exposed via the gastrointestinal tract. Furthermore, the continuing replication of FMDV in the oropharyngeal tonsils during viremia and peak clinical infection with no concurrent amplification of virus occurring in the lower respiratory tract indicates that these sites are the major source of shedding of FMDV from pigs.