报告题目: Coli phage 9g with modified guanine (dG+), DNA susceptibility to Type II restriction enzymes, and isolation of host mutants resistant to phage infection
报 告 人：Dr. Shuang-yong Xu
联 系 人：贺新义 firstname.lastname@example.org
E. coli phage 9g contains the modified base deoxyarchaeosine (dG+) in its genome. The phage encodes its own primase, DNA ligase, DNA polymerase, and enzymes necessary to synthesize and incorporate dG+. Here we report phage 9g DNA sensitivity to >200 Type II restriction endonucleases (REases). Among the REases tested approximately 29% generated complete or partial digestions, while the remaining 71% displayed resistance to restriction. Phage 9g restriction fragments can be degraded by DNA exonucleases or ligated by T3 and T4 DNA ligases. In addition, we examined a number of cytosine and adenine methyltransferases to generate double base modifications. M.AluI, M.CviPI, M.HhaI, and M.EcoGII were able to introduce 5mC or N6mA into 9g DNA as confirmed by partial resistance to restriction and by liquid chromatography-mass spectrometry. A number of wild-type E. coli bacteria restricted phage 9g, indicating natural restriction barriers exist in some strains. A BlastP search of GenBank sequences revealed five glutamine amidotransferase-QueC homologs in Enterobacteria and Pseudomonas phage, and distant homologs in other phage and bacterial genomes, suggesting that dG+ is not a rare modification. We also mapped phage 9g DNA packaging (pac) site containing two 21-bp direct repeats and a major terminase cut site in phage 9g genome.
Dr. Xu received his Ph.D. degree in Microbiology from University of Iowa and then worked as a postdoctoral associate at New England Biolabs (NEB) for two years. After that he has been working at NEB as a senior scientist for over 20 years. His research interests include structure and function studies of restriction enzymes and catalytic mechanism. He is also interested in engineering strand-specific nicking enzymes and artificial endonucleases, and in altering specificities of type IIB restriction enzymes. His new research directions are characterization of SauUSI-like (type IV) and type IIM BisI-family enzymes, and phage 9g with guanine modification.