Enzymes for DNA Manipulation in

Recombinant DNA

Enzymes for DNA Manipulation

•The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the test tube

•Depends on the availability of purified enzymes whose activities are known and can be controlled, and which can therefore be used to make specified changes to the DNA molecules that are being manipulated

•Within the cell these enzymes participate in essential processes: DNA replication and transcription breakdown of unwanted or foreign DNA repair of mutated DNA recombination between different DNA

molecules

• Four broad categories of the enzymes: DNA synthesis enzymes (DNA polymerases) Restriction enzymes (Nucleases ) Ligation enzymes (DNA Ligases) End-modification enzymes

https://www.youtube.com/watch?v=rhd_fBPyzSM

DNA synthesis enzymes:DNA Polymerases

DNA Polymerases•Play the central role in the processes of

life

•Enzymes that synthesize DNA and responsible of duplicating the genetic information

•Many of the techniques used to study DNA (PCR, DNA sequencing, DNA labelling etc) depend on the synthesis of copies of all or part of existing DNA or RNA molecules

Source: RCSB Protein Data Bank

DNA Polymerases : StructureEscherichia coli Thermus aquaticus

• Shaped roughly like a hand

• The space between the "fingers" and the "thumb" is just the right size for a DNA helix

• The template strand is coloured purple and the new strand is coloured green.

• The enzyme contains three separate active sites:

1. The polymerase site, near the top in this picture, synthesizes the new strand by adding nucleotides

2. The 3'-5' exonuclease site, near the center in the E. coli polymerase, proofreads the new additions

3. At the bottom is the 5'-3’ exonuclease site that later removes the small RNA fragments that are used to prime DNA replication

DNA Polymerases : Mode of action

•Synthesize new polynucleotides (DNA) complementary to an existing DNA or RNA template, via the base-pairing rules

•Template-dependent DNA polymerase: copy an existing DNA or RNA molecule

•Template-dependent DNA polymerase: can function only if the template possesses a double-stranded region which acts as a primer for initiation of polymerization

DNA synthesis requires a primer

The primer determines which part of a DNA molecule is copied

Primer: short synthetic oligonucleotide, usually about 20 nucleotides in length, which acts as a starting point for DNA synthesis

•Synthesized in the 5‘ → 3' direction

•DNA polymerase is the most accurate enzyme

• It creates an exact copy of your DNA each time, making less than one mistake in a billion bases

•DNA polymerase not only synthesizes new daughter strand DNA with high accuracy but also at an impressive pace; the DNA polymerase in mammals is capable of adding 50 nucleotides per second (!!) to the growing DNA chain

DNA replication and repair

DNA replication and repairA 3′→5′ exonuclease activity also called the proofreading activity because it allows the polymerase to correct errors by removing a nucleotide that has been inserted incorrectly

The types of DNA polymerases used in research

•Escherichia coli DNA polymerase I

•Thermostable DNA polymerase I

•Klenow DNA polymerase

•Reverse transcriptase (RNA-dependent DNA polymerase)

Escherichia coli DNA polymerase I

•Unmodified E. coli enzyme with 5‘ → 3' polymerase activity

•To initiate DNA synthesis there must be a short double-stranded region or a short synthetic oligonucleotide (primer) to provide a 3'-OH end onto which the enzyme will add new nucleotides.

• A 3' 5' exonuclease activity enables the enzyme to remove nucleotides from the 3' end of the strand that it has just synthesized

• This proofreading activity is for errors correction.

•5' 3' exonuclease activity to remove polynucleotide ahead.

DNA polymerase I

Nick translation (The 5´→ 3´ exonuclease activity removes nucleotides ahead of the growing DNA chain, allowing nick-translation)

Second-strand cDNA synthesis

Major uses of DNA Polymerase I from E. coli

*was used frequently in the early days of recombinant DNA technology for synthesizing cDNA. However, other enzymes have proven to be more effective , therefore this enzyme is no longer frequently used

Thermostable DNA polymerase I

•Like other DNA polymerases, catalyze template-directed synthesis of DNA

•Most common: Taq DNA polymerase I, obtained from bacteria Thermus aquaticus, which live in hot springs at temperatures up to 95 °C

•Taq Polymerase: Possess 5‘ →3' polymerase and 5' → 3' exonuclease activities

•Taq Polymerase: Lack 3' → 5' exonuclease activity (no proofreading)

Polymerase

3'->5'Exonuclease

Source and Properties

Taq No From Thermus aquaticus. Halflife at 95C is 1.6 hours

Pfu Yes

From Pyrococcus furiosus. Appears to have the lowest error rate of known thermophilic DNA polymerases

Vent Yes

From Thermococcus litoralis; also known as Tli polymerase. Halflife at 95 C is approximately 7 hours

Klenow DNA Polymerase

• Modified version of E. coli DNA polymerase I, by removing the 5' 3' exonuclease activity

• It retains the 5' 3' polymerase and 3' 5' exonuclease activities

Major uses• Filling in recessed 3’ ends

• Removing 3’ overhangs

Major uses of Klenow Polymerase

Both are used to create blunt ends on DNA fragments created by cleavage with restriction enzymes

• Second-strand cDNA synthesis. The lack of 5’ exonuclease activity offers a potential advantage for obtaining a maximum yield of long double-stranded cDNAs

• Labeling by random priming

• DNA sequencing by dideoxy method

• An enzyme that functions as a RNA-dependent DNA polymerase

• It makes DNA copies from RNA rather than DNA templates

• Encoded by retroviruses, where they copy the viral RNA genome into DNA prior to its integration into host cells

• Reverse transcriptases that are available commercially are derived from: (either by purification from the virus or expression in E. coli)

• Avian myeloblastosis virus (AMV)

• Moloney murine leukemia virus (M-MLV)

Reverse transcriptase

• Make DNA copies from mRNA molecules which is called complementary DNA (cDNA)

• Short primer are required to initiate the synthesis

Major uses of reverse transcriptase

RNA's polyadenylate tail (poly-A tail)polymers of thymidine (oligo dT primer)

dNTPs

cDNA

Other polymerases

•T4 DNA Polymerase very similar to Klenow DNA polymerase I

•T7 DNA Polymerase very similar to Klenow DNA polymerase I and

T4 mainly used in DNA sequencing by the chain

termination technique •Terminal Transferase

•Bacteriophage RNA Polymerase

Nucleases

Nucleases• Nuclease: any enzyme that cleaves nucleic acids,

degrade nucleic acid molecules by breaking the phosphodiester bonds

• Further divided into endonucleases and exonucleases

• Endonucleases are enzymes that cleave the phosphodiester bond within (endo) a polynucleotide chain

• Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain

•Restriction endonuclease

•Deoxyribonuclease I (DNase I)

•Ribonuclease A (RNase A)

The types of nucleases used in research

Restriction endonucleases (RE)

•Restriction endonucleases (restriction enzymes): play a central role in all aspects of recombinant DNA technology

•A restriction endonuclease is an enzyme that binds to a DNA molecule at a specific sequence and makes a double-stranded cut at or near that sequence

Types of RE

• Type I: complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences

• Type II: cut DNA at defined positions close to or within their recognition sequences

• Type III: cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests

• Type IV: recognize modified, typically methylated DNA

Patterns of DNA Cutting by RE: sticky ends or cohesive ends

• 5' overhangs: The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends

• 3' overhangs: asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3' ends

• Blunts: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs

Patterns of DNA Cutting by RE: blunt ends

https://www.youtube.com/watch?v=aA5fyWJh5S0

• Cleaves double-stranded or single stranded DNA

• Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues (=endonuclease)

• Some of the common applications of DNase I are: Eliminating DNA (e.g. plasmid) from preparations of

RNA Analyzing DNA-protein interactions via DNase

footprinting Nicking DNA prior to radiolabeling by nick

translation

Deoxyribonuclease I (DNase I)

• An endoribonuclease that cleaves single-stranded RNA at the 3' end of pyrimidine residues

• It degrades the RNA into 3'-phosphorylated mononucleotides and oligonucleotides

• Some of the major use of RNase A are: Eliminating or reducing RNA contamination in

preparations of plasmid DNA Mapping mutations in DNA or RNA by mismatch

cleavage. RNase will cleave the RNA in RNA:DNA hybrids at sites of single base mismatches, and the cleavage products can be analyzed

Ribonuclease A (RNase A)

• Exonuclease III (E. coli): Used most commonly to prepare a set of nested deletions of the termini of linear DNA fragments

• Nuclease S1 (Aspergillus): Used commonly to analyze the structure of DNA:RNA hybrids and to remove single-stranded extensions from DNA to produce blunt ends

• Mung Bean Nuclease (Mung bean sprouts): Used to remove single-stranded extensions from DNA to produce blunt ends

Other nucleases

Ligation enzymes: DNA Ligases

DNA Ligases•catalyze formation of a phosphodiester bond

between the 5' phosphate of one strand of DNA and the 3' hydroxyl of the adjacent DNA residues

•The natural role of DNA ligase is to repair single-strand breaks (nicks) in the sugar-phosphate backbone of a double-stranded DNA molecule

• Ligation requires energy, which is provided by adding either ATP or NAD

• The most widely used DNA ligase is obtained from E. coli cells infected with T4 bacteriophage: T4 DNA ligase

• During ligation, sticky ends increase the efficiency of ligation: Form base-pairing in ligation mixture Not stable but they persist for sufficient time for a

ligase enzyme to attach to the junction and synthesize phosphodiester bonds to fuse the ends together

Blunt ended, cannot form base pairing, the ligation is a much less efficient process

End of Lecture 3Thank You