LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19(lacO3/lacOs)), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOS and lacO3 were 5.7 +/- 0.3 x 10(-11) M and 4.1 +/- 0.2 x 10(-11) M respectively, which compare favorably with literature reports of 5 x 10(-10)-1 x 10(-9) M for native lacO1 and 1-1.2 x 10(-10) M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.     

Virion protein 16 induces demethylation of DNA integrated within chromatin in a novel mammalian cell model

DNA methylation and demethylation play important roles in mediating epigenetic regulation. So far, the mechanism of DNA demethylation remains elusive and controversial. Here, we constructed a plasmid, named with pCBS-luc, that contained an artificial CpG island, eight Gal4 DNA-binding domain binding site, an SV40 promoter, and a firefly luciferase reporter gene. The linearized pCBS-luc plasmid was methylated in vitro by DNA methyltransferase, and transfected into the HEK293 cells. The stable HEK293 transfectants with methylated pCBS-luc (me-pCBS-luc) were selected and obtained. The methylation status of the selected stable cell lines were confirmed by bisulfite sequencing polymerase chain reaction amplification. The methylation status could be maintained even after 15 passages. The virion protein 16 (VP16) was reported to enhance DNA demethylation around its binding sites of the promoter region in Xenopus fertilized eggs. Using our me-pCBS-luc model, we found that VP16 also had the ability to activate the expression of methylated luciferase reporter gene and induce DNA demethylation in chromatin DNA in mammalian cells. Altogether, we constructed a cell model stably integrated with the me-pCBS-luc reporter plasmid, and in this model we found that VP16 could lead to DNA demethylation. We believe that this cell model will have many potential applications in the future research on DNA demethylation and dynamic process of chromatin modification.     

Dynamics of DNA methylation pattern

Methylation patterns are the result of de novo methylation, demethylation, and the maintenance of existing methylation. Although the existence and identity of an active demethylase remain in doubt, recent evidence suggests that protein binding can specify sites of demethylation through a replication-dependent pathway. By using a stable episomal system in human cells, plus the Drosophila system, and mouse embryonic stem cells, we are beginning to understand the function and targets of de novo methyltransferases in murine and human cells.     

SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA–protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4–XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4–ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.     

The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study

LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex.     

大肠杆菌染色体上严谨型启动子的构建

【背景】启动子的渗漏表达是代谢工程和合成生物学较为关注的问题,探索严谨型启动子使之能像开关一样控制基因的表达有助于解决这一问题。【目的】为避免在质粒上研究启动子带来的弊端,本研究将在染色体上对严谨型启动子进行构建和评价。【方法】基于4种调控元件四环素tetO、乳糖lacO、阿拉伯糖araC和鼠李糖rhaR的序列,以及2种来源的启动子PL和Plac序列,设计和组合构建了6个启动子PtetO2、PtetO3、PlacO2、PlacO3、PlacO+ara和PlacO+rha。应用CRISPR/Cas9系统将这6个启动子序列整合到大肠杆菌ATCC 8739染色体上,利用绿色荧光蛋白(Green fluorescent protein,GFP)的表达,分析这6个启动子的相对表达强度和严谨型控制情况。【结果】GFP表达分析显示,启动子PlacO+rha为最佳严谨型启动子,在无诱导剂时表达为0.02,有诱导剂时最大表达强度为lac Z基因启动子的12倍,相对控制范围为600倍。【结论】研究结果将为代谢工程和合成生物学中的精确调控基因表达奠定良好的应用基础。     

Effect of CpG methylation on RAG1/RAG2 reactivity: implications of direct and indirect mechanisms for controlling V(D)J cleavage

It has been suggested that DNA methylation/demethylation is involved in regulating V(D)J rearrangement. Although methylated DNA is thought to induce an inaccessible chromatin structure, it is unclear whether DNA methylation can directly control V(D)J recombination independently of chromatin structure. In this study, we tested whether DNA methylation directly affects the reactivity of the RAG1/RAG2 complex. Specific methylation within the heptamer of the recombination signal sequences (RSS) markedly reduced V(D)J cleavage without inhibiting RAG1/RAG2–DNA complex formation. By contrast, methylation at other positions around the RSS did not affect the reactivity of the RAG proteins. The presence of a methyl-CpG binding-domain protein inhibited the binding of the RAG1/RAG2 complex to all the methylated CpG sites that were tested. Our findings suggest that DNA methylation around the RSS may have a previously unexpected function in regulating V(D)J recombination by directly inhibiting V(D)J cleavage, in addition to its general function of inducing an inaccessible chromatin configuration.     

Expression of cholecystokinin type A receptor gene correlates with DNA demethylation during postnatal development of rat pancreas.

Cholecystokinin stimulates pancreatic amylase secretion, gallbladder contraction, and pancreatic growth, etc. by binding with high affinity to a cholecystokinin type A receptor (CCKAR). To better understand the expression of CCKAR mRNA in terms of tissue specificity and postnatal development, we determined the methylation status of BssHII sites (5'-B sites) in the rat CCKAR gene promoter. The 5'-B sites in adult pancreas expressing CCKAR mRNA were much less extensively methylated than those in fetal pancreas not expressing the mRNA. In brain, liver, and kidney of adult rats not expressing CCKAR mRNA, the 5'-B sites were methylated. In pancreas, the demethylation level of the sites increased at 21 days after birth. Concomitant with the DNA demethylation level in the 5'-B sites, the mRNA level rose rapidly in 21 days. These results demonstrate that methylation and expression of the CCKAR gene reveal a good inverse correlation.