5’-Phosphorylation Increases the Efficacy of Nucleoside Inhibitors of the DNA Repair Enzyme SNM1A

Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5’–3’ exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5’-position, as well as zinc-binding groups at the 3’-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5’-phosphate dramatically increased the potency of the inhibitors.     

DNA光修复酶的功能及其在化妆品中的应用

本文介绍了紫外线对皮肤伤害的基本原理,对DNA光修复酶在抵抗紫外线损伤中的重要功效进行了综述,并介绍了其作为化妆品功能成分开发的现状,预测其作为基因修复型抗日晒产品的广阔应用前景。     

The Emerging Roles of ATP-Dependent Chromatin Remodeling Enzymes in Nucleotide Excision Repair

DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo.     

化学核酸酶的合成、表征及与pBR322DNA的作用

合成了三角架配体L,用红外光谱、核磁共振氢谱、X单晶衍射等手段对配体进行了鉴定。在此基础上,用高氯酸镁与其合成镁的金属络合物,通过核磁共振氢谱、红外光谱等手段对配合物的结构进行了表征。最后,通过电泳技术研究了此配合物与pBR322DNA的作用,并对机理进行了初步探讨。电泳结果表明：配合物在生理条件下(pH=7．0,37℃)与pBR322DNA反应2h,能够断裂DNA,有的甚至断裂成线性。本工作的意义在于：配合物在生理条件下,能迅速水解DNA,甚至出现了线性断裂产物。     

Synthesis of a stabilized version of the imidazolone DNA lesion

Imidazolone (dIz) is an abundant, highly mutagenic, and rather unstable DNA lesion that can cause dG-->dC transversion mutations. dIz is generated in DNA by a variety of oxidative processes such as type I photooxidation. Herein we report the synthesis of a carbocyclic nucleoside analogue of dIz and of DNA containing this stabilized lesion analogue. The carbocyclic modification protects this lesion analogue from anomerization. As the repair of the lesion analogue by DNA glycosylases is not possible, this analogue should allow cocrystallization studies together with wild-type repair enzymes. Characterization of the lesion analogue was performed by using spectroscopic methods and enzymatic digestion experiments of the oligonucleotides.     

Novel Diazirine‐Containing DNA Photoaffinity Probes for the Investigation of DNA‐Protein‐Interactions

An investigation of the precise interactions between damaged DNA and DNA repair enzymes is required in order to understand the lesion recognition step, which is one of the most fundamental processes in DNA repair. Most recently, photoaffinity labeling approaches have enabled the analysis of even transient protein‐DNA interactions. Here we report the synthesis and evaluation of oligonucleotides that contain two photoaffinity “catcher moieties” next to incorporated DNA lesions. With these DNA constructs it is possible to analyze the interactions between DNA lesions and the appropriate repair enzymes. The probes labeled the repair protein efficiently enough to enable subsequent protein analysis by mass spectrometry.     

Differential Inhibitory Activities and Stabilisation of DNA Aptamers against the SARS Coronavirus Helicase

The helicase from severe acute respiratory syndrome coronavirus (SARS‐CoV) possesses NTPase, duplex RNA/DNA‐unwinding and RNA‐capping activities that are essential for viral replication and proliferation. Here, we have isolated DNA aptamers against the SARS‐CoV helicase from a combinatorial DNA library. These aptamers show two distinct classes of secondary structure, G‐quadruplex and non‐G‐quadruplex, as shown by circular dichroism and gel electrophoresis. All of the aptamers that were selected stimulated ATPase activity of the SARS‐CoV helicase with low‐nanomolar apparent K_m values. Intriguingly, only the non‐G‐quadruplex aptamers showed specific inhibition of helicase activities, whereas the G‐quadruplex aptamers did not inhibit helicase activities. The non‐G‐quadruplex aptamer with the strongest inhibitory potency was modified at the 3′‐end with biotin or inverted thymidine, and the modification increased its stability in serum, particularly for the inverted thymidine modification. Structural diversity in selection coupled to post‐selection stabilisation has provided new insights into the aptamers that were selected for a helicase target. These aptamers are being further developed to inhibit SARS‐CoV replication.     

Terminal Protection of Small Molecule-Linked DNA for Small Molecule–Protein Interaction Assays

Methods for the detection of specific interactions between diverse proteins and various small-molecule ligands are of significant importance in understanding the mechanisms of many critical physiological processes of organisms. The techniques also represent a major avenue to drug screening, molecular diagnostics, and public safety monitoring. Terminal protection assay of small molecule-linked DNA is a demonstrated novel methodology which has exhibited great potential for the development of simple, sensitive, specific and high-throughput methods for the detection of small molecule–protein interactions. Herein, we review the basic principle of terminal protection assay, the development of associated methods, and the signal amplification strategies adopted for performance improving in small molecule–protein interaction assay.