Enzymes for manipulating DNA *** Buffers and solution conditions*** I. DNA polymerases III. Kinase and alkaline phosphatase IV. Nucleases V. Topoisomerase

Buffers are crucial for activity of enzymes! Ideal biochemical buffers: pKa between 6 and 8 Chemically inert Polar (soluble and not membrane permeable) Non-toxic Inexpensive Salt and temperature indifferent

Enzyme “reaction buffers”: Buffer: Tris, HEPES, etc. Salt: NaCl, KCl, PO4-, etc.--stabilizes protein structure, facilitates protein-DNA interactions Divalent metal ions: Mg2+, Ca2+, Zn2+, etc.--often required for enzyme activity Glycerol: (for storage)--stabilizes protein structure EDTA: chelates (removes) divalent cations--important especially for storage, if your enzyme is especially sensitive to metal ion-dependent proteases Beta mercaptoethanol or dithiothreitol: reducing agents that prevent illegitimate disulfide bond formation Non-specific protein: Bovine serum albumin (BSA) Other cofactors, eg. ATP, NADH

DNA polymerases--making copies, adding labels, or fixing DNA E. coli DNA polymerase I --the classic DNA polymerase Moderately processive polymerase 3'->5' proof-reading exonuclease 5'->3' strand-displacing (nick-translating) exonuclease Used mostly for labelling DNA molecules by nick translation.

DNA polymerases Native T7 DNA polymerase --highly processive, with highly active 3'->5' exonuclease Useful for extensive DNA synthesis on long, single-stranded (e.g. M13) templates Useful for labeling DNA termini and for converting protruding ends to blunt ends Modified T7 polymerase (Sequenase) --lack of both 3'->5' exonuclease and 5'->3' exonuclease Ideal for sequencing, due to high processivity Efficiently incorporates dNTPs at low concentrations, making it ideal for labeling DNA In molecular biology and biochemistry, processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate". For example,processivity is the average number of nucleotides added by a polymerase enzyme, such as DNA polymerase, per association event with the template strand.

DNA polymerases Reverse transcriptase RNA-dependent DNA polymerase Essential for making cDNA copies of RNA transcripts Cloning intron-less genes Quantitation of RNA

Terminal transferase template-independent DNA polymerase Incorporates dNTPs onto the 3' ends of DNA chains Useful for adding homopolymeric tails or single nucleotides (can be labelled) to the 3' ends of DNA strands (make DNA fragments more easily clonable).

T4 polynucleotide kinase Transfers gamma phosphate of ATP to the 5’ end of polynucleotides Useful for preparing DNA fragments for ligation (if they lack 5’ phosphates) Useful for radiolabelling DNA fragments using gamma 32P ATP as a phosphate donor

alkaline phosphatase Catalyzes removal of 5’ (and 3’) phosphates from polynucleotides Useful for treating restricted vector DNA sequences prior to ligation reactions, prevents religation of vector in the absence of insert DNA Lack of vector 5’ phosphates may inhibit transformation efficiency? Use only when absolutely necessary…

Nucleases Exonucleases Endonucleases Remove nucleotides one at a time from a DNA molecule Endonucleases Break phosphodiester bonds within a DNA molecule Include restriction enzymes

Exonucleases Bal 31 Double-stranded exonuclease, operates in a time-dependent manner Degrades both 5’ and 3’ ends of DNA Useful for generating deletion sets, get bigger deletions with longer incubations.

Exonucleases Exonuclease III--double-stranded DNA 3’-5’ exonuclease activity 3’ overhangs resistant to activity, can use this property to generate “nested” deletions from one end of a piece of DNA (use S1 nuclease to degrade other strand of DNA) Aspergillus nuclease S1 is an endonuclease enzyme derived from Asperligus oryzae that splits ssDNA and RNA into oligo- or mononucleotides. This enzyme catalyzes the following reaction Endonucleolytic cleavage to 5'-phosphomononucleotide and 5'-phosphooligonucleotide end-products

Exonucleases Exonuclease I 3’-5’ exonuclease Works only on single-stranded DNA Useful for removing unextended primers from PCR reactions or other primer extension reactions

Endonucleases Dnase I Cleaves double-stranded DNA randomly (also cleaves single-stranded DNA) Mn++: both strands of DNA cut Mg++: single strands nicked Very useful for defining binding sites for DNA binding proteins

Types of endonucleases Type I: multisubunit proteins that function as a single protein complex, usually contain two R subunits,two M subunits and one S subunit Type II: recognize specific DNA sequences and cleave at constant positions at or close to that sequence to produce 5’-phosphates and 3’-hydroxyls. Most useful in cloning!! Type III: composed of two genes (mod and res) encoding protein subunits that function either in DNA recognition and modification (Mod) or restriction (Res) Type IV: one or two genes encoding proteins that cleave only modified DNA, including methylated, hydroxymethylated and glucosyl-hydroxymethylated bases

Topoisomerase A restriction enzyme and ligase--all in one altering the “linking number” in coiled, constrained (supercoiled) DNA--relaxing DNA twisting during replication

Topoisomerase Topoisomerase catalyzed ligation is EXTREMELY efficient (>85% of resulting plasmids are recombinant)--excellent for library constructions Can be used to clone blunt ended DNA (PCR products, restriction digests), T-overhang PCR products (from Taq polymerase), and directional clones