第二章 Enzymology of Genetic Engineering.ppt

Lesson 5 Exonuclease Exonucleases are enzymes (found as individual enzymes, or as parts of larger enzyme complexes) that cleave nucleotides one at a time from an end of a polynucleotide chain. These enzymes hydrolyze phosphodiester bonds from either the 3 or 5 terminus of a polynucleotide molecule. 1. The definition of exonuclease 2. The characteristics of commonly used exonucleases Exonuclease Substrate Cut site Product E.coli exo I ssDNA 5’-OH terminus 5’ mononucleotide, dinucleotide E.coli exo III dsDNA 3’-OH terminus 5’ mononucleotide E.coli exo V DNA 3’-OH terminus 5’ mononucleotide E.coli exo VII ssDNA 3’-OH terminus, 5’-P terminus 2-12bp oligonucleotide λexo dsDNA 5’-P terminus 5’ mononucleotide T7 gene 6 exonuclease dsDNA 5’-P terminus 5’ mononucleotide Table 2.4 The characteristics of commonly used exonucleases Lesson 5 Single-Strand Specific Endonuclease S1 nuclease (S1核酸酶) is an endonuclease that is active against single-stranded DNA and RNA molecules. It is five times more active on DNA than RNA. Although its primary substrate is single-stranded, it can also occasionally introduce single-stranded breaks in double-stranded DNA or RNA, or DNA-RNA hybrids. It is used in the laboratory as a reagent in nuclease protection assays (核酸酶保护分析). In Molecular biology it is used in removing single stranded tails from DNA molecules to create blunt ended molecules and opening hairpin loops generated during synthesis of double stranded cDNA (see Fig 2.29). 1. S1 nuclease Fig. 2.32 Intron/exon location by nuclease protection assay Fig. 2.33 Detecting gene mutation by nuclease protection assay BAL-31 nulease is an endonuclease specific for single-stranded nucleic acids (activity I). It also has exonuclease activity (activity II), and degrades double-stranded nucleic acids from both the 3- and 5-ends when single-stranded nucleic acids are not present. BAL-31 can be used to shorten progressive of double-stranded DNA fragments at both termini, or to draw