Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

用PFU法研究微型生物群集过程中数据的处理

根据MacArthur-Wilson的岛屿区系平衡模型S_t=S_(eq)(1-e~(GT)),可以从野外生态效应试验和室内毒性试验中,提出3个功能参数(S_(eq)、G、t_(90％))进行比较。本文提出两种计算方法:复合梯形法和最小二乘法,后者已在计算机上实现了BASIC计算程序。从数学理论上论证,最小二乘法误差较小,但如果实验布局合理,两种计算方法能得到十分一致的结果。实验模型是否符合理论模型,可以用统计学上的拟合差异度检验法来检验。     

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca^2 +, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R₂C₂) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Molecular characterization of adeno-associated viruses infecting children

Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (approximately 98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.     

Computational characterization of the sequence landscape in simple protein alphabets

We characterize the "sequence landscapes" in several simple, heteropolymer models of proteins by examining their mutation properties. Using an efficient flat-histogram Monte Carlo search method, our approach involves determining the distribution in energy of all sequences of a given length when threaded through a common backbone. These calculations are performed for a number of Protein Data Bank structures using two variants of the 20-letter contact potential developed by Miyazawa and Jernigan [Miyazawa S, Jernigan WL. Macromolecules 1985;18:534], and the 2-monomer HP model of Lau and Dill [Lau KF, Dill KA. Macromolecules 1989;22:3986]. Our results indicate significant differences among the energy functions in terms of the "smoothness" of their landscapes. In particular, one of the Miyazawa-Jernigan contact potentials reveals unusual cooperative behavior among its species' interactions, resulting in what is essentially a set of phase transitions in sequence space. Our calculations suggest that model-specific features can have a profound effect on protein design algorithms, and our methods offer a number of ways by which sequence landscapes can be quantified.     

Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses

Human replication protein A (RPA), a heterotrimeric protein complex, was originally defined as a eukaryotic single-stranded DNA binding (SSB) protein essential for the in vitro replication of simian virus 40 (SV40) DNA. Since then RPA has been found to be an indispensable player in almost all DNA metabolic pathways such as, but not limited to, DNA replication, DNA repair, recombination, cell cycle, and DNA damage checkpoints. Defects in these cellular reactions may lead to genome instability and, thus, the diseases with a high potential to evolve into cancer. This extensive involvement of RPA in various cellular activities implies a potential modulatory role for RPA in cellular responses to genotoxic insults. In support, RPA is hyperphosphorylated upon DNA damage or replication stress by checkpoint kinases including ataxia telangiectasia mutated (ATM), ATR (ATM and Rad3-related), and DNA-dependent protein kinase (DNA-PK). The hyperphosphorylation may change the functions of RPA and, thus, the activities of individual pathways in which it is involved. Indeed, there is growing evidence that hyperphosphorylation alters RPA-DNA and RPA-protein interactions. In addition, recent advances in understanding the molecular basis of the stress-induced modulation of RPA functions demonstrate that RPA undergoes a subtle structural change upon hyperphosphorylation, revealing a structure-based modulatory mechanism. Furthermore, given the crucial roles of RPA in a broad range of cellular processes, targeting RPA to inhibit its specific functions, particularly in DNA replication and repair, may serve a valuable strategy for drug development towards better cancer treatment.