AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg(2+), Ca(2+), Sr(2+) and Ba(2+) were used for clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg(2+), Ca(2+) and Sr(2+) are counterions. When Ba(2+) is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B-A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr(2+) induces the greatest structural transition.     

Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is unlocked by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.     

Measurements of Cry1F binding and activity of luminal gut proteases in susceptible and Cry1F resistant Ostrinia nubilalis larvae (Lepidoptera: Crambidae)

The biochemical mechanism of resistance to the Bacillus thuringiensis Cry1F toxin was studied in a laboratory-selected strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) showing more than 3000-fold resistance to Cry1F and limited cross resistance to other Cry toxins. Analyses of Cry1F binding to brush border membrane vesicles of midgut epithelia from susceptible and resistant larvae using ligand immunoblotting and Surface Plasmon Resonance (SPR) suggested that reduced binding of Cry1F to insect receptors was not associated with resistance. Additionally, no differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Considering these results along with previous evidence of relatively narrow spectrum of cross resistance and monogenic inheritance, the resistance mechanism in this Cry1F selected strain of O. nubilalis appears to be specific and may be distinct from previously identified resistance mechanisms reported in other Lepidoptera.     

Allotransplantation of adult spinal cord tissues after complete transected spinal cord injury: Long-term survival and functional recovery in canines

Science China Life Sciences - Spinal cord injury (SCI), especially complete transected SCI, leads to loss of cells and extracellular matrix and functional impairments. In a previous study, we...     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

紫外线辐射对西伯利亚鲟精子活力和寿命的影响

研究了不同剂量紫外线辐射(254nm,UVC)对西伯利亚鲟精子活力和寿命的影响.结果表明:紫外线辐射对精子的活力、快速运动时间和寿命均具有显著性影响.其中,精子活力随辐射剂量的增加而呈先迅速下降,后迅速上升,再迅速下降的趋势;精子快速运动时间的变化趋势与活力相似;精子寿命随辐射剂量的增加呈缓慢下降的趋势.当辐射剂量达288mJ.cm-2时,精子无快速运动,当辐射剂量达324mJ.cm-2时,精子活力和寿命均降为0.根据Hertwig效应判断,辐射剂量216mJ.cm-2为西伯利亚鲟精子灭活的最适剂量.     

Efficient production of <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed roots and composite plants in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Recalcitrance of most large-seeded legumes, such as peanut, to regeneration and genetic transformation has hampered studies on gene function and efforts for genetic improvement. Agrobacterium rhizogenes-mediated transformation provides a system for rapid and efficient transformation of plant tissues. In this study, embryonic axes along with cotyledons of peanut were injected with a suspension culture of A. rhizogenes using microliter syringes. The influence of several factors such as plant genotype, A. rhizogenes culture stage, co-culture period of A. rhizogenes, and acetosyringone concentration in the co-cultivation medium have been evaluated. It is found that A. rhizogenes-mediated transformation of peanut is genotype-independent. Up to 61% transformation was recorded when embryonic axes were co-cultivated with 5 × 10⁷ A. rhizogenes cells from logarithmic phase for 2 days on co-culture medium containing 50 μmol l⁻¹ acetosyringone. Composite plants with transgenic roots were harvested after 45 days of treatment. Furthermore, this method was applied to assess the insecticidal activity of a synthetic cry8Ea1 gene against Holotrichia parallela in transgenic roots of peanut.     

Exonuclease Function of Human Mre11 Promotes Deletional Nonhomologous End Joining

DNA double-stranded breaks (DSBs) are lethal if not repaired and are highly mutagenic if misrepaired. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways and can rejoin the DSB ends either precisely or with mistakes. Recent evidence suggests the existence of two NHEJ subpathways: conservative NHEJ (C-NHEJ), which does not require microhomology and can join ends precisely; and deletional NHEJ (D-NHEJ), which utilizes microhomology to join the ends with small deletions. Little is known about how these NHEJ subpathways are regulated. Mre11 has been implicated in DNA damage response, thus we investigated whether Mre11 function also extended to NHEJ. We utilized an intrachromosomal NHEJ substrate in which DSBs are generated by the I-SceI to address this question. The cohesive ends are fully complementary and were either repaired by C-NHEJ or D-NHEJ with similar efficiency. We found that disruption of Mre11 by RNA interference in human cells led to a 10-fold decrease in the frequency of D-NHEJ compared with cells with functional Mre11. Interestingly, C-NHEJ was not affected by Mre11 status. Expression of wild type but not exonuclease-defective Mre11 mutants was able to rescue D-NHEJ in Mre11-deficient cells. Further mutational analysis suggested that additional mechanisms associated with methylation of Mre11 at the C-terminal glycine–arginine-rich domain contributed to the promotion of D-NHEJ by Mre11. This study provides new insights into the mechanisms by which Mre11 affects the accuracy of DSB end joining specifically through control of the D-NHEJ subpathway, thus illustrating the complexity of the Mre11 role in maintaining genomic stability.DNA double-stranded breaks (DSBs)³ can be produced in physiological and genotoxic processes. Improper repair or failure to repair DSBs can lead to gene deletions, duplications, translocations, and missegregation of large chromosome fragments, which may result in gene dosage imbalance, cancer development, or cell death (1–3). Historically, two distinct pathways have been described which ensure that DSBs are repaired: nonhomologous end joining (NHEJ) and homologous recombination (HR). During HR, the damaged chromosome interacts via synapsis with an undamaged DNA molecule with which it shares extensive sequence homology, usually its sister chromatid (4, 5). HR is most active in the late S and G₂ phases of the cell cycle. In contrast, NHEJ is active throughout the cell cycle and requires little or no DNA homology during repair; thus, it is traditionally considered an error-prone repair pathway (6, 7). However, accumulating evidence from recent studies suggests that there exists an error-free NHEJ subpathway (8, 9).Two types of end-joining reactions can be defined operationally. The first one, which may be called conservative NHEJ (C-NHEJ), is characterized by the precise joining of short, overhanging, complementary ends. Proteins including Ku70/Ku80 and XRCC4 (10–12) are associated with this highly efficient pathway, whereby most ends are rejoined successfully without any alteration of the DNA sequence (8). The alternative pathway for NHEJ is the highly mutagenic and deletional NHEJ (D-NHEJ), which results in short deletions after use of imperfect microhomology of about 1–10 bp at the repair junctions. D-NHEJ activity has been demonstrated in the budding yeast Saccharomyces cerevisiae. In addition, D-NHEJ is independent of Rad52, Rad1, or Ku80 but depends on Mre11 in yeast (13, 14). However, the genetic determinants of this subpathway have not been well established in mammalian cells.Mre11 is the core subunit of the Mre11·Rad50·Nbs1 complex (called the MRN complex), which is conserved throughout all kingdoms of life. The MRN complex is a central player in most aspects of the cellular response to DSBs, including HR, NHEJ, telomere maintenance, and DNA damage checkpoints (15–17). Loss of Mre11 results in increased radiosensitivity and chromosomal instability (17). Patients with germ line mutations of Mre11 have clinical presentations similar to those of ataxia telangiectasia patients (ataxia telangiectasia-like disorder) (18).After DNA damage, the MRN complex is recruited to the sites of damage via zinc hooks at the ends of the long, flexible arms of Rad50 (19, 20). Mre11 contains both single-stranded DNA endonuclease and 3′-5′ exonuclease activities in vitro, but in vivo Mre11 is also implicated in 5′-3′ DSB resection. The MRN complex also interacts with BRCA1 and CtIP, which may be essential for DSB end resection to generate 3′ overhanging single-stranded DNA during initiation of HR (21, 22).Mre11 has an N-terminal nuclease domain, which contains five phosphoesterase motifs, and a C-terminal glycine–arginine-rich domain (GAR). Arthur et al. (23) showed that an H85L mutation completely abrogated exonuclease activity, whereas binding to Rad50 and Nbs1 was retained. Complementation of ataxia telangiectasia-like disorder cells with this mutant, called Mre11-3, restored the localization of the MRN complex to DSBs in IR-induced foci (23, 24). Methylation of the GAR region has also been shown to be important for the DNA binding and exonuclease activity of Mre11 in vitro (25, 26). Both the crystal structure of yeast Mre11 and data from conditional knock-out mice (Mre11^H129N/Δ) reveal that the nuclease activity of Mre11 is required for HR repair of DSBs (22, 27). However, the role of Mre11 in NHEJ is not well defined (27, 28). Most recently, Mre11 was reported to support NHEJ in mammalian cell (29–31). However, whether Mre11 regulates both NHEJ subpathways or only D-NHEJ is controversial, and the mechanisms by which Mre11 is involved in NHEJ remain to be established.To address these questions, we have established a system that can analyze the accuracy and efficiency of rejoining of two adjacent DSB ends at chromosomal level in human embryonic kidney 293 (HEK293) cells. We show here that Mre11 siRNA knockdown in these cells results in significant reduction of the overall NHEJ efficiency. Upon sequencing the repair junctions, we found that Mre11 siRNA knockdown suppressed D-NHEJ by ∼10-fold, reflected by a reduction of small deletions in the repair junction, but it had no effect on the efficiency of C-NHEJ. Mutation of Mre11 in either the phosphoesterase domain (Mre11-3) or the GAR region (Mre11-R/A) to produce abnormal exonuclease activity impaired the D-NHEJ pathway only. The D-NHEJ deficiency is significantly more severe in cells with Mre11-R/A than that in cells with Mre11-3. Therefore, our data suggest that Mre11 is required specifically for D-NHEJ repair of DNA DSBs and that its exonuclease activity is at least one of the important mechanisms for this DNA end joining subpathway.