Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Naturally occurring abasic sites in DNA exist as an equilibrium mixture of the aldehyde, the hydrated aldehyde, and the hemiacetal forms (dominant). The influence of the configuration of the C1' hydroxyl group of the hemiacetal form on duplex structure and abasic site repair has been examined using novel carbocyclic analogues. Both the alpha- and beta-forms of this novel abasic site were introduced into oligomeric DNA using the standard DMT-phosphoramidite approach in an automated solid-phase synthesizer. Solution structures of the d(CGTACXCATGC).d(GCATGAGTACG) duplex (where X is the alpha- or beta-anomer of the carbocyclic abasic site analogue) were determined by NMR spectroscopy and restrained molecular dynamics simulations. The structures were only minimally perturbed by the presence of either anomer of the abasic site. All residues adopted an anti conformation, and Watson-Crick alignments were observed on all base pairs of the duplexes. At the lesion site, the abasic residues and their partner adenines showed increased dynamic behavior but adopted intrahelical positions in the final refined structures. Incision of duplexes having the alpha- or beta-anomer of the carbocyclic abasic site by human AP endonuclease showed that the enzyme recognizes both configurations of the lesion and nicks the DNA backbone with similar efficiency. Our results challenge the suggestion that Ape1 is stereoselective and imply a plasticity at the active site of the enzyme for accommodating either anomer of the lesion.
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PMID:Impact of the C1' configuration of abasic sites on DNA duplex structure. 1558 47

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a variety of malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma (NPC). Functions of most EBV genes have not been determined. The use of bacterial artificial chromosome (BAC) to clone and modify the genome of EBV has enhanced the gene function study in the context of genome. Infectious clones of EBV were previously established by using EBV-BAC plasmid p2089. In order to further investigate EBV mutant biology, an easy and efficient method for gene modification in EBV-BAC was developed and detailed. The kanamycin gene (kan) flanked by recombinase FLP recognition targets (FRTs) was amplified from plasmid pKD13 and inserted into the vector of pcDNA3.1(+). Through the introduction of restriction endonuclease BsmB I in PCR primers, NPC-derived LMP1 gDNA containing the full-length ORF was then precisely ligated with kan on pcDNA3.1(+). The linear DNA segment of kan-LMP1 was transformed into E. coli DH10B cells containing p2089 and plasmid pKD46, homologous recombination was subsequently mediated by redalphabetagamma system from bacteriophage lambda. By this linear transformation and ET cloning, the full-length LMP1 in EBV-BAC (p2089) was replaced by the kan-LMP1. The introduced kan gene in EBV-BAC genome was eliminated specifically by the recombinase FLP when transformed by plasmid pCP20, leaving an FRT scar of 69 bp. The mutant could be identified by antibiotic screening and PCR amplification on bacteria medium. This method allows the gene of interest to be easily modified alone and then to be introduced into EBV-BAC genome. Following this example of gene substitution, other mutations such as deletion, insertion and point mutation become convenient work, and this improved method can be a potential use of gene modification in other BAC-based herpesvirus genome.
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PMID:[Gene modification in the genome of Epstein-Barr virus cloned as a bacterial artificial chromosome]. 1847 68