bt 403+mod iii nkj+lecture 1 dna+modifying+enzymes 2011

7/28/2019 BT 403+MOD III NKJ+Lecture 1 DNA+Modifying+Enzymes 2011

1/24

BT-403 MOD-III NKJ LECTURE : DNA MODIFYING ENZYMES :2011

Restriction Enzyme

OVERVIEW

"Restriction enzymes were discovered about 30 years ago during investigations into thephenomenon of host-specific restriction and modification of bacterial viruses. Bacteria

initially resist infections by new viruses, and this "restriction" of viral growth stemmed

from endonucleases within the cells that destroy foreign DNA molecules. Among the firstof these "restriction enzymes" to be purified were EcoR I and EcoR II from Escherichia

coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes were found

to cleave DNA at specific sites, generating discrete, gene-size fragments that could be re-

joined in the laboratory. Researchers were quick to recognize that restriction enzymesprovided them with a remarkable new tool for investigating gene organization, function

and expression."

(http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asp)

RESTRICTION ENZYME TYPES

"Restriction enzymes are traditionally classified into three types on the basis of subunitcomposition, cleavage position, sequence-specificity and cofactor-requirements.However, amino acid sequencing has uncovered extraordinary variety among restriction

enzymes and revealed that at the molecular level there are many more than three different

kinds.

Type I enzymes are complex, multisubunit, combination restriction-and-modificationenzymes that cut DNA at random far from their recognition sequences. Originally
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asphttp://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asp


2/24

thought to be rare, we now know from the analysis of sequenced genomes that they are

common. Type I enzymes are of considerable biochemical interest but they have little

practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.

Type II enzymes cut DNA at defined positions close to or within their recognition

sequences. They produce discrete restriction fragments and distinct gel banding

patterns, and they are the only class used in the laboratory for DNA analysis and genecloning. Rather then forming a single family of related proteins, type II enzymes are a

collection of unrelated proteins of many different sorts. Type II enzymes frequently differ

so utterly in amino acid sequence from one another, and indeed from every other knownprotein, that they likely arose independently in the course of evolution rather than

diverging from common ancestors.

The most common type II enzymes are those like Hha I, Hind III and Not I that cleave

DNA within their recognition sequences. Enzymes of this kind are the principle onesavailable commercially. Most recognize DNA sequences that are symmetric because they

bind to DNA as homodimers, but a few, (e.g., BbvC I: CCTCAGC) recognize

asymmetric DNA sequences because they bind as heterodimers. Some enzymes

recognize continuous sequences (e.g., EcoR I: GAATTC) in which the two half-sites ofthe recognition sequence are adjacent, while others recognize discontinuous sequences

(e.g., Bgl I: GCCNNNNNGGC) in which the half-sites are separated. Cleavage leaves a 3

-hydroxyl on one side of each cut and a 5-phosphate on the other. They require onlymagnesium for activity and the corresponding modification enzymes require only S-

adenosylmethionine. They tend to be small, with subunits in the 200350 amino acid

range.

BOB by "Weird Al" Yankovic

The next most common type II enzymes, usually referred to as type IIs" are those like

Fok I and Alw I that cleave outside of their recognition sequence to one side. These

enzymes are intermediate in size, 400650 amino acids in length, and they recognize

sequences that are continuous and asymmetric. They comprise two distinct domains, onefor DNA binding, the other for DNA cleavage. They are thought to bind to DNA as

monomers for the most part, but to cleave DNA cooperatively, through dimerization of

the cleavage domains of adjacent enzyme molecules. For this reason, some type IIsenzymes are much more active on DNA molecules that contain multiple recognition sites.

The third major kind of type II enzyme, more properly referred to as "type IV" are

large, combination restriction-and-modification enzymes, 8501250 amino acids in

length, in which the two enzymatic activities reside in the same protein chain. Theseenzymes cleave outside of their recognition sequences; those that recognize continuous

sequences (e.g., Eco57 I: CTGAAG) cleave on just one side; those that recognize

discontinuous sequences (e.g., Bcg I: CGANNNNNNTGC) cleave on both sidesreleasing a small fragment containing the recognition sequence. The amino acid
http://www.youtube.com/watch?v=Nej4xJe4Tdghttp://www.youtube.com/watch?v=Nej4xJe4Tdg


3/24

sequences of these enzymes are varied but their organization are consistent. They

comprise an N-terminal DNA-cleavage domain joined to a DNA-modification domain

and one or two DNA sequence-specificity domains forming the C-terminus, or present asa separate subunit. When these enzymes bind to their substrates, they switch into either

restriction mode to cleave the DNA, or modification mode to methylate it.

Type III enzymes are also large combination restriction-and-modification enzymes.

They cleave outside of their recognition sequences and require two such sequences inopposite orientations within the same DNA molecule to accomplish cleavage; they rarely

give complete digests. No laboratory uses have been devised for them, and none are

available commercially."(http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asp)

NAMING RESTRICTION ENZYMES

Restriction enzymes are named based on the bacteria in which they are isolated in the

following example for the enzyme EcoRI:

E Escherichia (genus)co coli (species)

R RY13 (strain)I First identified Order ID'd in bacterium

TYPES OF ENDS GENERATED FROM DIGEST

There are three types of ends generated from restriction digests, blunt ends (no

overhangs), 5' overhangs, and 3' overhangs.
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asphttp://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asp


4/24

The same types of ends can be generated by different enzymes. These enzyme pairs are

said to generate "compatible ends" because the overhangs can hydrogen bond with each

other and base pair.

FREQUENCY OF ENZYME RECOGNITION

The frequency of restriction enzyme recognition depends on the number of nucleotides

used in the site recognition. Each position of the recognition site has four possible bases

and therefore the probability of finding any one base is 1/4. To determine the probabilityof finding a particular sequence, you need to multiply the probability of each site by all of

the other sites. If the length of the recognition site is 4 then the probability of finding that

site is 1/4 * 1/4 * 1/4 * 1/4 = 1/256. For a site with six bases in the recognition site theprobability would be (1/4)^6 = 1/4096. The reciprocal of these probabilties tell us that we

would expect to see cuts in a completely random sequence, on the average, every 4096

bases for a six base recognition enzyme. Now, since most DNA sequences are notcompletely random, we do no always see a normal distribution of fragment lengths

around this estimated size.


5/24

CUTTING CONDITIONS

Most restriction enzymes function adequately at pH 7.4. All type II enzymes require

Mg2+ and vary in their requirement for ionic strength (usually provided by NaCl).Proteins are further stabilized by addition of a strong reductant such as dithiothreitol

(DTT). Some restriction enzymes are sentitive to protein dilution and are benefited by theaddition of non-enzymatic proteins such as bovine serum albumin (BSA).

Restriction enzyme stocks are most often shipped with a 10X reaction buffer that isoptimized for the enzyme. Most of these reaction buffers can be classified into four

general categories, based on their ionic strength:

0 mM NaCl = low salt buffer (L)

50 mM NaCl = medium salt buffer (M)

100 mM NaCl = hi salt buffer (H)

150 mM NaCl = very high salt buffer (VH)

When digesting with two or more enzymes in the same buffer that do not use the same

buffer, it is important to consult activity tables, such as 3.1.2, which list the relative

activities in the various classes of restriction enzyme concentrations. For example, if a

double digest was being made with EcoRI (hi salt) and HpaII (low salt, KCl), Table 3.1.2tells us that both enzymes are active in medium salt. Since HpaII requires KCl instead of

NaCl, you would use a medium concentration KCl buffer.

When some restriction enzymes find themselves in suboptimal conditions they cutabnomally. For example, EcoRI, when cutting in low salt buffers does not just recognize

its normal sequence G^AAATTC but will also recognize ^AATT sites. This kind of

activity is called *Star activity. You must therfore be careful of reaction conditions when

dealing with enzymes that have known star activity.

Most restriction enzyme reactions can be run at 37C. There are some enzymes,

however, that have other optimal temperatures (see table 3.1.1).

ENZYME REACTIONS

By convention, enzyme activity is defined in terms of units, where 1 unit is the quantityof enzyme that will completely cut all sites on 1 g of DNA in 1 hour. Since not all DNAsequences contain the same number of sites for an enzyme, the DNA that is used for

definition of the unit scale must also be included. There are some common viral and

plasmid DNAs that are commonly used to define the enzyme unit, such as lambda, SV40,and pBR322.


6/24

Restriction enzymes are shipped and stored in 50% glycerol to stabilize the protein and

prevent freezing, which often denatures protein and inactivates enzymes. Enzyme

function is inhibited by high concentrations of glycerol > 5% and so stock enzymes mustbe diluted at least 1/10 in reaction mixtures in order to prevent glycerol inhibition.

Since there is not a set number of enzyme sites per length of every DNA, whendigesting a DNA for the first time it is a good idea to double the amount of enzyme and

double the time of the digest in order to ensure complete digestion. The quality or purityof the DNA being digested can greatly impact its digestability. Restriction enzyme

digests on DNA that is contaminated with proteins, excessive RNA, or base

modifications can be hard to digest, requiring either longer digestion times, increasedenzyme, or both.

Reduced digestion time and the dilution of enzyme can each be used to create partial

digestions of target DNA. The best case senerio for a partial digest will produce a set of

fragments that are randomly cleaved at each of the restriction enzyme sites. In reality, not

all restrictions sites are cut with equal probability and so not all fragment sizepermutations may be represented at the same concentration.

Digestion of a large amount of DNA may be cost prohibitive at the 1U/1g DNA

concentration. You can use lower amounts of enzyme / DNA if the incubation times ofthe digest are extended. A good rule is to never use less than 0.5 units of enzyme in a

digest. Many restriction enzymes become inactive when protein concentrations are

reduced below this level. The dilution effect can sometimes be counteracted by addingBSA to keep overall protein concentrations high. Also, not all restriction enzymes are

active for extended periods of time. Check the manufacturer data for stability information

if you are planning extended incubations.

Restriction enzymes are stored at -20C. When working with enzymes outside the

freezer, make sure that you keep them on ice and return them to the freezer as soon

as possible. When setting up a restriction enzyme digest, always add the restriction

enzyme last so that it has the proper buffer and approximate reaction conditions.

A typical restriction enzyme digest can be set up as follows:

ComponentStock

ConcentrationVolume stock/reaction

DNA (0.5 - 1 g) up to 16 L

10X Buffer 2 L = 1/10 total volume

(BSA, if needed) 100X 0.2 L = 1/100 total volume

nanopure wateras needed to bring total reaction

volume to 20 L

Restriction Enzyme

(=>0.5 U)5-50 Units up to 2 L


7/24

Total = 20 L

Note that there is flexibility in the amount of DNA and the corresponding amount of

water that you can add to the reaction. You should be warned, if you choose to add alarge volume of a dilute DNA to the reaction and the DNA is contaminated with proteins

or RNA, the reaction may be inhibited. Take care, when adding large volumes of DNA,to make sure that the DNA is pure.

STOPPING RESTRICTION ENZYME DIGESTIONS

After the incubation period is complete, digestions may be stopped by adding EDTA,

which chelated Mg2+. Some enzymes may be inactivated by heating (see table 3.1.1) but

others may require extraction with a protein denaturant such as phenol/chloroform orseparation of the protein from the DNA on a column (Qiagen and others).

SEPARATION OF RESTRICTION ENZYME FRAGMENTS BY LENGTH

DNA fragments that vary in size can be separated by electrophoresis in an agarose or

polyacrylamide gel.

DNA bands in the gel can be visualized by adding ethidium bromide or other DNA

binding dye such as SYBR green. Exposure of the gel to UV light will cause the bounddye to fluoresce and thus allow the DNA to be visualized and the relative positions

photographed.


8/24

The size of the DNA fragments can be estimated by comparison to the relative

migration of DNA standards of known size.

Note that the plot of migration vs size produces a curve. The curve can be linearized by

plotting the distance migrated against the log(DNA size). A linear regression analysis onlog transformed data can be used to determine the quality of the data and estimate the size

and associated errors of unknown fragments.

POLYMERASES

DNA Polymerase I - DNA-dependent DNA plymerase with inherent 3'->5'

exonuclease activity (proofreading) and 5'->3' exonuclease activity (nick translation).Good for copying short stretches of DNA.


9/24

Large Klenow Fragment DNA Polymerase I - DNA polymerase I without the 5'-3'activity.

Klenow Fragment DNA Polymerase I - Large Klenow fragment with mutation that

also eliminated the 3'->5' exonuclease activity.

T7 DNA Polymerase - highly processive DNA polymerase capable of copying longstretches of DNA. Modified forms reduce the very strong 3'->5' exonuclease activity.


10/24

Taq DNA Polymerase - a therostable DNA polymerase that possesses a 5'->3'

polymerase activity and a double strand specific 5'->3' exonuclease activity. (No

proofreading).

Vent DNA Polymerase - A thermostable 5'->3' DNA polymerase with 3'->5'exonuclease activity (proofreading). Also available in a proofreading minus form for

DNA sequencing.

Reverse Transcriptase - An RNA directed DNA polymerase that uses RNA or DNAas a template and makes complementary DNA by extending an RNA or DNA primer.

Lacks 3'->5' exonuclease activity (proofreading) .


11/24

Terminal transferase - a template independent polymerase that add dNTPs to the 3'

OH of DNA (protruding, recessed, blunt-ended, double- or single-stranded DNA.

Sp6 RNA Polymerase - RNA Polymerase that has a highly specific recognition site,CATACGATTTAGGTGACACTATAG.

T7 RNA Polymerase - RNA Polymerase that has a highly specific recognition site,CTCGAGTAATACGACTCACTATAGG.

T3 RNA Polymerase - RNA Polymerase that has a highly specific recognition site,

ATTAACCCTCACTAAAGGGA.

Poly(A) Polymerase - Adds As onto the end of single-stranded RNA.

DNA Ligase:


12/24

The cloning of DNA fragments into plasmid, phage, or other vectors requires the

ligation of the DNA fragments to the vector. This requires the enzyme DNA Ligase.

T4 DNA ligase is isolated from the T4 bacteriophage and is commercially available.

There are also several other commercially available sources of ligase which use ATP as

an energy source. Ligase isolated from E.coli uses NAD as an energy source instead ofATP.

As illustrated above, the ligase enzymes require that a adjacent bases have a 5'

phosphate and a 3' hydroxyl group to serve as substrates.

T4 DNA Ligase can ligate both blunt or sticky ended DNA.


13/24

The efficiency of ligation is much higher for sticky ends than for blunt ends. For bluntends we need about 10X more ligase and lower temperatures (12-14C, less thermally

efficient than 20C) in order to get ligation.

Exercise:

1. If you have a fragment, I, cut with EcoRI, that you want to insert into a linearized

vector, V cut with EcoRI, and you mix equimolar amounts of each with ligase, what areall of the possible ligation products?

2. If you wanted to increase the percent of vector that contained insert, what would you

change?

3. How does molar concentration impact self-ligation vs intramolecular ligation?

Addition of Linkers to blunt-ended DNA

If you have a fragment with blunt ends that you want to clone, you can improve the

efficiency of ligation by adding short linkers that contain a restriction enzyme site.

Usually the linker is present in a 100X molar concentration to the target fragment toensure that all ends receive a linker. With this excess of linker, it is highly probable that

more than one linker will be added. This is resolved by cleaving the excess linkers off

with the restriction enzyme specific for the site that the linkers carry.


14/24

Addition of adapters to blunt-ended DNA

An alternative, to avoid cutting up the target, is to use adapters. Adapters are designed

to ligate at the blunt end but not at the sticky end (no 5' P).

Producing sticky ends with homopolymer tailing

The enzyme terminal deoxynucleotidyl transferase (or terminal transferase for short)

adds nucleotides tothe 3' end of DNA without requiring a template.


15/24

If only a single nucleotide triphosphate is available to the enzyme, then it will add a

homopolymer tail to the 3' ends of linear DNA. By adding complementary tails to thefragment to be inserted and the vector, a fragment of DNA can be inserted into a vector.

The advantage of this system is that the vector will not ligate back together without an

insert. Since the complementary homopolymer tails are most likely not the same length,the klenow fragment of DNA polymerase will need to be added along with the ligase in

order to repair nicks before ligation.

NUCLEASES

S1 nuclease - Exonuclease that degrade both 3' and 5' termini of duplex DNA.

Endonuclease cuts at nicks and gaps and single-stranded regions.


16/24

Mung Bean nuclease - a single-stranded specific DNA and RNA endonuclease which

will degrade single-stranded extensions from the ends of DNA and RNA leaving blunt,ligatable ends.

Bal 31 nuclease - Exonuclease that degrade both 3' and 5' termini of duplex DNA.

Endonuclease cuts at nicks and gaps and single-stranded regions.

Exo III nuclease - 3' exonuclease that acts at blunt or 3' recessed ends. Is not active on

on single-stranded DNA (i.e., 3' overhangs => 4 bases).


17/24

Exo I nuclease - Degrades single-stranded DNA primers from annealed PCR products.

Lambda exonuclease - highly processive enzyme that acts in the 5'->3' direction,

catalyzing the removal of 5' mononucleotides from duplex DNA. 5' phosphrylated DNAgreatly preferred . It is unable to cut DNA at nick or gaps.


18/24

DNase I - nonspecifically cleaves DNA to release di, tri, and oligonucleotide products

with 5' P and 3' OH .

RNase I - cuts all single-stranded RNA bonds leaving a 5' -OH and a 2', 3' cyclicmonophosphate.

RNase H - endonuclease which specifically hydrolyzes RNA which is hybridized toDNA. It does not digest single- or double-stranded DNA.


19/24

OTHER DNA MODIFYING ENZYMES

Alkaline Phosphatase(AP) - catalyzes the removal of 5' phosphates from DNA,

RNA, and NTPs or dNTPs.

Polynucleotide Kinase (PNK) - catalyzes the transfer and exchange of Pi from thegamma position of ATP to the 5' hydroxyl terminus of double- and single-stranded DNA

and RNA


20/24

DNA Ligases

T4 DNA Ligase - Catalyzes the formation a phosphodiester bond between justaposed5' phosphate and 3' hydroxyl group in duplex DNA or RNA. Will join blunt or cohesive

ends.


21/24

E.coli DNA Ligase - Catalyzes the formation a phosphodiester bond between

justaposed 5' phosphate and 3' hydroxyl group in duplex DNA containing cohesive ends.

DNA methylases

dam methylase - methylates A at: TA(me)TC

Topoisomerases

Topoisomerase I (E. coli) - relaxes negatively supercoiled DNA.

RECOMBINASES

Cre Recombinase - A type I topoisomerase that catalyzes the site-specific

recombination of DNA between loxP sites.

RESTRICTION ENZYME MAPS


22/24

Maps of restriction enzyme cleavage sites are useful in planning not only cloning projects but

also in gene identification, forensics, sequencing and mutagenesis projects.

Below is a description and examples of three methods used to organize restriction enzyme

location on DNA fragments.

ESTIMATING FRAGMENT SIZES FROM RE MAP

There are both linear and circular maps.

We will start by estimating, from the maps, the fragment sizes that would be generated

when cutting with particular enzyme of combinations of enzymes.If you cut a linear DNA you will get one more fragment than you have RE sites.

When cutting circular DNA, you get the same number of fragments as there are RE sites.

Example, given the map below, find the sizes generated with the RE combinations:

Restriction Enzyme Fragment Sizes

A 600, 1000

B 400, 800

C 200, 1400

A + B 200, 400, 600

A + C 200, 400, 1000B + C 200, 400, 600

Try the circular map:


23/24

Restriction Enzyme Fragment Sizes

A 400, 1200

B 1600

C 200, 1400

A + B 400, 600

A + C 200, 400, 800

B + C 200, 1200

COMPLETE DIGESTION OF UNKNOWN DNA

When mapping RE by complete digestion, often it is best to use information from both single

and double digests.

Start with the simplest cuts first, for example the Bgl and Kpn single digests and compare them

to the Bgl + Kpn double digest. Keep one of the single digest fixed (Bgl) and adjust the map

fragments for the other single and double digest until both singles and the double digest cut sitesalign. Repeat this for each of the other single/double digest sets until the map of the RE sites are

consistent throughout.

COMPLETE DIGESTION OF DNA CLONED INTO A KNOWN VECTOR

When a fragment of DNA with an unknown restriciton map is cloned into a vector containing

many flanking restriction enzyme sites, it is fairly easy to identify the location of most single cuts

within the unknown portion.

For example, if a fragment cut with Cla I was inserted into a 2000 bp vector with the following

multiple cloning site:


24/24

---- Eco RI --Pst I -- Cla I -- Bam HI -- Hind III ------

and subsequent cutting with each of the flanking enzymes created the following fragments,

what RE map could you create?

Eco RI - 450, 2550

Pst I - 200, 2600

Bam HI - 3000

Hind III - 500, 2500

Answer:

-EcoRI-PstI-ClaI-(200)-PstI-(200)-PstI-(50)-EcoRI-(50)HindIII-(500)ClaI-BamHI-HindIII ---

PARTIAL DIGEST OF END LABELED DNA

Partial digest sites can be mapped from the labeled end. If the 5' end is labeled, then begin the

map with the smallest fragments being located at the 5' end. If the 3' end is labeled then start themap with the largest fragments being located at the 5' end.

bt 403+mod iii nkj+lecture 1 dna+modifying+enzymes 2011

Documents