Damage site chromatin: open or closed?

Technical advances in recent years, such as laser microirradiation and chromatin immunoprecipitation, have led to further understanding of DNA damage responses and repair processes as they happen in vivo and have allowed us to better evaluate the activities of new factors at damage sites. Facilitated by these tools, recent studies identified the unexpected roles of heterochromatin factors in DNA damage recognition and repair, which also involves poly(ADP-ribose) polymerases (PARPs). The results suggest that chromatin at damage sites may be quite structurally dynamic during the repair process, with transient intervals of 'closed' configurations before a more 'open' arrangement that allows the repair machinery to access damaged DNA.     

Bumps in the road: how replicative DNA polymerases see DNA damage

Significant advances have been made recently in the study of polymerases. First came the realization that there are many more DNA polymerases than originally thought; indeed, no fewer than 14 template-dependent DNA polymerases are found in mammals. Concurrent structural studies of DNA polymerases bound to DNA and incoming nucleotide have revealed how these remarkable copying machines select the correct deoxynucleoside triphosphate among a sea of nucleotides. A whole new level of insight into DNA replication fidelity has been reached as a result of recently determined crystal structures of DNA lesions in the context of the active sites of repair, replicative and specialized DNA polymerases. These structures illustrate why some lesions can be bypassed readily, whereas others are strong blocks to DNA replication.     

真核细胞中基因的转录调控

叙述了真核细胞三种RNA聚合酶合成的基因的转录调控.由于真核细胞DNA含量非常大,其基因的转录调控具有以下特点：参与的转录因子多;与顺式DNA序列元件结合呈一定顺序.这反映了真核细胞中基因的转录调控是由多个转录因子间的相互作用来实现的．     

In vitro production and screening of DNA polymerase eta mutants for catalytic diversity

Mutant DNA polymerases have become an increasingly important tool in biotechnology. The ability to examine the activity and specific properties of enzymes has a crucial role in the characterization of the enzyme. We have developed several systems for characterizing DNA polymerases that combine random mutagenesis with in vivo selection systems. However in vivo screening systems for specific properties are sometimes unavailable. The ability to quickly screen for polymerase activity has many applications, including the identification of compounds that can inhibit polymerase activity, identifying the properties of newly discovered polymerases, and engineering new biological properties into existing polymerases. These applications can both expand the knowledge of the basic science of polymerases and can further industrial efforts to identify new drugs that specifically target polymerase activity. Here we present a high-throughput in vitro assay to select for active polymerases. We show the applicability of this assay by measuring the level of activity for a set of in vitro synthesized polymerase mutants and by screening for the incorporation of a fluorescent nucleotide analog by DNA polymerases.     

植物基因工程中人工启动子的研究进展

启动子是调控基因转录的一段DNA,也是构建基因工程表达载体的重要元件。天然启动子在表达强度和特异性等方面存在一定的局限性。采用人工构建的方法,有望得到诱导因子广、本底活性低、表达强度高、启动表达快等特点的启动子。本文综述了人工启动子在诱导表达、组织特异性表达、高效表达等方面的研究进展。     

Design,activity, and structure of a highly specific artificial endonuclease

We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.