Summary Enzymology of Recombinant DNA Summary Jiyoun Lee

Summary : Enzymology of Recombinant DNA [Summary] Jiyoun Lee Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 1

Summary : Enzymology of Recombinant DNA 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 [http: //biotech. snu. ac. kr] 2

Summary : Enzymology of Recombinant DNA 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 [http: //biotech. snu. ac. kr] 3

Summary : Enzymology of Recombinant DNA Enzymes that break DNA and RNA backbone bonds • Endonucleases – Restriction enzymes : Three types – Deoxyrebinucleases (DNase) • DNase I and Mung Bean Nuclease – Ribonucleases (RNase) • RNase T 1, U 2, A, CL 3, Phy. M, B, H • Exonucleases – – – Exonuclease III Exonuclease VII Lambda Exonuclease T 7 Gene 6 Exonuclease Venom phosphodiesterase Spleen phosphodiesterase Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 4

Summary : Enzymology of Recombinant DNA • Endo- and Exonuclease – – Nuclease Bal 31 Neurospora Crassa Nuclease P 1 Nuclease S 1 Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 5

Summary : Enzymology of Recombinant DNA 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 ds. DNA 4000~7000 bp – Type II : twofold symmetry in ds. DNA – Type III : specific pentameric or hexameric cognate sequence in ds. DNA but cleavage 25~27 nucleotides to 3’ side Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 6

Summary : Enzymology of Recombinant DNA 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 [http: //biotech. snu. ac. kr] 7

Summary : Enzymology of Recombinant DNA 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 [http: //biotech. snu. ac. kr] 8

Summary : Enzymology of Recombinant DNA Endo- and Exonuclease • Nuclease Bal 31 – Highly specific single-stranded endodeoxynuclease activity – Exonuclease activity capable of simultaneously degrading both 3’ and 5’ termini of duplex DNA • Neurospora Crassa Nuclease – On ss. DNA or RNA : acts as an endonuclease – On ds. DNA (and ss. DNA) : acts as an exonuclease Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 9

Summary : Enzymology of Recombinant DNA Enzymes that mend DNA and RNA backbone bonds • E. coli DNA Ligase • T 4 RNA Ligase Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 10

Summary : Enzymology of Recombinant DNA Diagramatic representation of the mechanism of action of bacterial DNA ligase Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 11

Summary : Enzymology of Recombinant DNA Enzymes that synthesize new DNA and RNA backbone bonds • • • DNA Polymerase I Large fragment DNA polymerase I (Klenow fragment) T 4 DNA polymerase Modified T 7 DNA polymerase Taq DNA polymerase RNA polymerases polymerase – Bacterial RNA polymerase – Bacteriophage RNA polymerase (T 3, T 7, and SP 6) • • Reverse transcriptase Poly(A) polymerase Terminal deoxynucleotidyl transferase Polynucleotide phosphorylase Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] transferase 12

Summary : Enzymology of Recombinant DNA Enzymes that add or remove phosphate at nucleic acid termini • T 4 polynucleotide kinase • Alkaline phosphatase • Tobacco acid pyrophosphatase Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 13

Summary : Enzymology of Recombinant DNA Enzymes and proteins that protect, coat, twist and untwist DNA • DNA methylase • Single-stranded nucleic acid binding proteins – Rec. A protein, SSB, Gene 32 protein • Topoisomerases Cell and Microbial Engineering Laboratory [http: //biotech. snu. ac. kr] 14