Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 1 Enzymology of Recombinant DNA [Summary] Jiyoun Lee

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 2 Enzyme Definition –A protein that speeds up chemical reactions in the body. –Any of a group of chemical substances which are produced by living cells and which cause particular chemical reactions to happen while not being changed themselves. How it works? –Stabilize the transition state –Lower the activation energy What can we do? –Manipulation of nucleic acids (DNA, RNA)

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 3 Classification Enzymes that break DNA and RNA backbone bonds Enzymes that mend DNA and RNA backbone bonds Enzymes that synthesize new DNA and RNA backbone bonds Enzymes that add or remove phosphate at nucleic acid termini Enzymes and proteins that protect, coat, twist and untwist DNA

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 4 Enzymes that break DNA and RNA backbone bonds Endonucleases –Restriction enzymes : Three types –Deoxyrebinucleases (DNase) DNase I and Mung Bean Nuclease –Ribonucleases (RNase) RNase T1, U2, A, CL3, PhyM, B, H Exonucleases –Exonuclease III –Exonuclease VII –Lambda Exonuclease –T7 Gene 6 Exonuclease –Venom phosphodiesterase –Spleen phosphodiesterase

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 5 Endo- and Exonuclease –Nuclease Bal31 –Neurospora Crassa Nuclease –Nuclease P1 –Nuclease S1

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 6 Restriction enzyme Definition –A degregative enzyme that recognizes and cuts up DNA at specific 'cut sites' called restriction sites. Three types –Type I : random cleaving at unmethylated dsDNA 4000~7000 bp –Type II : twofold symmetry in dsDNA –Type III : specific pentameric or hexameric cognate sequence in dsDNA but cleavage 25~27 nucleotides to 3’ side

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 7 DNase and RNase DNase I –Degrades DNA by hydrolyzing internal phosphoester linkages Mung Bean Nuclease –Highly specific for DNA or RNA lacking an ordered structure –5’  3’ RNase H –Specifically degrades the RNA strands in DNA:RNA heteroduplexes

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 8 Exonuclease Exonuclease III –3’  5’ exonuclease activity and other three activites –Manifested with double-stranded but not single-stranded DNA Exonuclease VII –3’  5’ or 5’  3’ direction –Single-strand specific exonuclease –Does not release mononucleotides Lambda Exonuclease –5’  3’ direction –Double-stranded exonuclease

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 9 Endo- and Exonuclease Nuclease Bal31 –Highly specific single-stranded endodeoxynuclease activity –Exonuclease activity capable of simultaneously degrading both 3’ and 5’ termini of duplex DNA Neurospora Crassa Nuclease –On ssDNA or RNA : acts as an endonuclease –On dsDNA (and ssDNA) : acts as an exonuclease

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 10 Enzymes that mend DNA and RNA backbone bonds E.coli DNA Ligase T4 DNA Ligase T4 RNA Ligase

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 11 Diagramatic representation of the mechanism of action of bacterial DNA ligase

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 12 Enzymes that synthesize new DNA and RNA backbone bonds DNA Polymerase I Large fragment DNA polymerase I (Klenow fragment) T4 DNA polymerase Modified T7 DNA polymerase Taq DNA polymerase RNA polymerases –Bacterial RNA polymerase –Bacteriophage RNA polymerase (T3, T7, and SP6) Reverse transcriptase Poly(A) polymerase Terminal deoxynucleotidyl transferase Polynucleotide phosphorylase polymerase transferase

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 13 Enzymes that add or remove phosphate at nucleic acid termini T4 polynucleotide kinase Alkaline phosphatase Tobacco acid pyrophosphatase

Cell and Microbial Engineering Laboratory [ Summary : Enzymology of Recombinant DNA 14 Enzymes and proteins that protect, coat, twist and untwist DNA DNA methylase Single-stranded nucleic acid binding proteins –RecA protein, SSB, Gene 32 protein Topoisomerases