ENZYMES THAT MODIFY DNA AND RNA1. RESTRICTION ENDONUCLEASES AND

METHYLASES• RESTRICTION ENDONUCLEASES EXIST IN NATURE IN

PROKARYOTES• Prokaryotic cells have restriction modification systems and will

cleave foreign DNA that enters the bacteria cell (e.g. bacteriophage) but not host DNA that has been protected or modified by methylation

• source of enzyme reagents, essential for generating recombinant DNA molecules

• Need to understand how they work in order to avoid problems when manipulating recombinant DNA

TYPES OF RESTRICTION ENDONUCLEASESThere are 3 types; Type I, II and IIITypes I and III contain the restriction and modification activities in the

same multiunit enzyme complex• Require ATP for cleavage• cleave DNA a substantial distance from the recognition sequence• not commonly usedType II• RE are not physically associated with methylases• do not require ATP for cleavage• generally cleave within or very near the recognition sequence• isolated 100's of different type II REs, many of which are available

commercially• The first type II RE characterised was from E.coli and was

designated E.coRI• EcoRI binds to DNA region with a specific palindromic sequence

of 6 bp and cuts between the G and the A residues on each strand

• It specifically cleaves the internucleotide bond between the oxygen of the 3'C of the sugar of one nucleotide and the phosphate group attached to the 5' carbon of the sugar of the adjacent nucleotide

NAMING R.E.• A 3 letter abbreviation based on the genus and species of

bacteria e.g. Eco = E.coli• a 4th letter can be used to indicate strain eg Hind• Roman numerals are used to designate the order of

characterisation of the different R.E. from the same organism• e.g. HpaI and HpaII- the first and second R.E. isolated from

Haemophilus parainfluenzaeRECOGNITION SITES• The palindromic sequences where most type II R.E. bind and cut

a DNA molecule are called recognition sites• Recognition sites of many type II R.E. contain 4-6 specific

nucleotidesCLEAVAGE• Can result in sticky ends or blunt ends

Enzymes; Practical considerations

• Expensive• Many are cloned recombinant enzymes but still can be expensive• One unit of a Restriction enzyme is defined as the amount that will

cut 1ug of a test DNA in 1h at optimum temp

• Rate of cutting is dependent on1. Number of sites/ug DNA2. linear, circular or supercoiled DNA3. R.E. sites near ends may not cut well4. Contaminated DNA may not cut well5. More enzyme required if buffer conditions are not optimum6. Ability to cleave depends on surrounding sequence

Enzymes; Practical considerations cont………

• Manufacturers catalogues give optimum buffers, temps and stabilities

• If xs enzyme is used may result in non specific cutting (called star activity)

• Contamination of enzyme stocks is disasterous. Use clean tips all the time.

• Enzymes should be stored in -20C freezer(not frost free)

• Enzymes should be placed on ice immediately on removal from freezer

• Enzymes should be used immediately and then returned to freezer

• Diluted enzymes are generally unstable. Do not dilute for long term storage

• Wear gloves to prevent contaminating enzymes with proteases and RNases

often present on fingers

ENZYMES THAT MODIFY DNA OR RNA cont.

2. Polymerases

The purpose of all polymerases is to join single nucleotides into a polymer

5’ to 3’ polymerase activity

•All DNA polymerases use deoxynucleotide 5’ triphosphates (dNTPs)

•Removes 2 phosphate groups (releasing a pyrophosphate and using

the released energy) from NTP and attaches the newly exposed 5’

phosphate to the 3’ hydroxyl of another nucleotide, generating a

phosphodiester bond

•Most polymerases require a template

•Most require a primer

Polymerases can have other activities as well as polymerase (building) activity:

3’ to 5’ exonuclease activityMany polymerase have this activity, useful for proof readingRemoves single mismatchesCombination of 5’ to 3’ polymerase and 3’ to 5’ exonuclease activity is particularly useful for making blunt ends and labeling 3’ ends

5’ to 3’ exonuclease activityOnly some polymerases have this activityUseful for removing RNA templates for nick translation

Ribonuclease H activityPresent in a few polymerasesDegrades RNA in RNA/DNA complexes

Properties of polymerases• Turnover number-nucleotides/min• Processivity-how many nucleotides added before

disassociates• Error frequency- how frequently generates a

mismatch(#errors/base pair)• Errors are dependent on conditions, pH, conc dNTP,

divalent cations• Every polymerase makes a mistake about 1 in

100000bp. Usually caught and proofread. The proofread error frequency is 1/1000000, making an overall error frequency of 1 in 10 billion bp)

Examples

1. DNA dependant DNA polymerase: E.coli polymerase 1

Acts primarily as proofreader (both 3 to 5 and 5 to 3 exonuc act and

polym act.)

Has RNase H act to degrade RNA primers

Plays role in replication

2. DNA dependant RNA polymerase: RNA polymerase

Transcribes ssRNA from dsDNA in transcription

Polymerase cont.

3. RNA dependant DNA polymerase: Reverse transcriptaseMakes DNA from RNA templates also has RNase H activity and can destroy the RNA in an RNA DNA hybrid molecule

Polymerases cont.

4. Template independent polymerase: terminal deoxynucleotide transferase (TdT)No templateUseful for generating restriction sites at blunt ends and labelling

Polymerase3'->5'

ExonucleaseSource and Properties

Taq NoFrom Thermus aquaticus. Halflife at

95C is 1.6 hours.

Pfu YesFrom Pyrococcus furiosus. Appears to

have the lowest error rate of known thermophilic DNA polymerases.

Vent YesFrom Thermococcus litoralis; also

known as Tli polymerase. Halflife at 95 C is approximately 7 hours.

Thermo tolerant polymerases used for PCR (polymerase chain reactions) reactionsThe total error rate of Taq polymerase has been variously reported between 1 x 10-4 to 2 x 10-5 errors per base pair. Pfu polymerase appears to have the lowest error rate at roughly 1.5 x 10-6

error per base pairVent is intermediate between Taq and Pfu.

Polymerases cont

Kinase

3. Kinase – catalyses the transfer of the gamma phosphate group of ATP to the 5’

hydroxyl of polynucleotide (all phosphates have to be removed from end). By combining a Phosphatase with a kinase the 5’ end of DNA can be labeled with a labeled phosphate group.

• e.g. Polynucleotide Kinase

• It is a product of the T4 bacteriophage, and commercial preparations are usually products of the cloned phage gene expressed in E. coli. The enzymatic activity of PNK is utilized in two types of reactions:

• PNK transfers the gamma phosphate from ATP to the 5' end of a polynucleotide (DNA or RNA). The target nucleotide is lacking a 5' phosphate either because it has been dephorphorylated or has been synthesized chemically.

• In the "exchange reaction", target DNA or RNA that has a 5' phosphate is incubated with an excess of ADP - in this setting, PNK will first transfer the phosphate from the nucleic acid onto an ADP, forming ATP and leaving a dephosphorylated target. PNK will then perform a forward reaction and transfer a phosphate from ATP onto the target nucleic acid.

Kinase reactions

Phosphatases

4. Phosphatase- catalyses the hydrolysis of 5’ phosphate groups from DNA or RNA or single nucleotides. Often used to prevent relegation of plasmids once they have been opened by restriction digest (since ligase requires a 5’ phosphate for ligation )

e.g. Alkaline phosphatase removes 5' phosphate groups from DNA and RNA. It will also remove phosphates from nucleotides and proteins. These enzymes are most active at alkaline pH

There are several sources of alkaline phosphatase that differ in how easily they can be inactivated:

• Bacterial alkaline phosphatase (BAP) is the most active of the enzymes, but also the most difficult to

• Calf intestinal alkaline phosphatase (CIP) most widely used in molecular, less active than BAP, but it can be effectively destroyed by protease digestion or heat

• Shrimp alkaline is readily destroyed by heat (65C for 15 minutes). Primary uses for alkaline phosphatase in DNA manipulations: • Removing 5' phosphates from plasmid and bacteriophage vectors

and preventing self-ligation • Removing 5' phosphates from fragments of DNA prior to labeling

with labelled phosphate.

5. DNA ligases catalyze formation of a phosphodiester bond between the 5' phosphate of one strand of DNA and the 3' hydroxyl of the another to permit joining of 2 DNA molecules together

• e.g. The most widely used DNA ligase is derived from the T4 bacteriophage. T4 DNA ligase requires ATP as a cofactor. It also requires ds DNA.

• T4 RNA ligase can use ssRNA or ssDNA substrates

1- Ligation of DNA with complementary cohesive termini

Ligase

Ligase continued

2- Repair reaction• H bonds are not enough to hold sticky ends together. A means of

reforming the internucleotide linkage between 3’OH and 5’phosphate groups is required and ligase does this

Nucleases6. Nucleases: DNase and RNaseMost of the time nucleases are evil when you are trying to preserve the

integrity of RNA or DNA samples. Many types differing in substrate specificity, cofactor requirements, and

whether they cleave nucleic acids internally (endonucleases), chew in from the ends (exonucleases) or attack in both of these modes.

The most widely used nucleases are DNase I and RNase A Deoxyribonuclease I cleaves double-stranded or single stranded DNA. • Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues• an endonuclease. • Major products are 5'-phosphorylated di, tri and tetranucleotides. • In the presence of magnesium ions, DNase I hydrolyzes each strand of

duplex DNA independently, generating random cleavages. • In the presence of manganese ions, the enzyme cleaves both strands of

DNA at approximately the same site, producing blunt ends or fragments with 1-2 base overhangs.

• DNase I does not cleave RNA 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 labeling by nick translation.

Nucleases cont.

Ribonuclease A is 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.