Enzymology of Recombinant DNA [Summary]

1. Enzymology of Recombinant DNA [Summary] Jiyoun Lee

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)

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 removephosphate at nucleic acid termini • Enzymes and proteins that protect, coat, twist and untwist 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

5. Endo- and Exonuclease • Nuclease Bal31 • Neurospora Crassa Nuclease • Nuclease P1 • Nuclease S1

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

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

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

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

10. Enzymes that mend DNA and RNA backbone bonds • E.coli DNA Ligase • T4 DNA Ligase • T4 RNA Ligase

11. Diagramatic representation of the mechanism of action of bacterial DNA ligase

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

13. Enzymes that add or remove phosphate at nucleic acid termini • T4 polynucleotide kinase • Alkaline phosphatase • Tobacco acid pyrophosphatase

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