Engineering site-specific endonucleases.
Site-specific endonucleases are involved in many aspects of the biochemistry of nucleic acids. Restriction enzymes and their relatives have become paradigms for enzymes acting on DNA. Numerous efforts have been devoted to changing their specificity by rational protein design, with, by and large, little success, presumably because the recognition process is highly redundant and recognition and catalysis are tightly coupled. This chapter describes one of the few successful examples of a change in specificity--namely the conversion of the mismatch repair nuclease, MutH, which, when stimulated by MutS and MutL, nicks hemimethylated d(GATC) sites, into a variant that cleaves fully methylated DNA as well as hemimethylated and unmethylated DNA. This chapter will describe the various steps involved in this design project, starting from the analysis of the structure and the identification of candidate amino acid residues responsible for sensing the methylation status, to the generation and characterization of MutH variants with an altered specificity toward hemimethylated d(GATC) sites.
Friedhoff
Peter
P
Institut für Biochemie, Justus-Liebig-Universität, Giessen, Germany.
Pingoud
Alfred
A
D000595:Amino Acid Sequence; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D045643:DNA Repair Enzymes