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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
blaber@sb.fsu.edu


Lecture 4

DNA modifying enzymes

Methylases, Polymerases, Nucleases, Ligases, Kinase, Phosphatase

Methylases

Just as the study of the bacterial restriction-modification sytem has provided a variety of specific endonucleases, there are also available a variety of specific DNA methylases.

Methylation of DNA usually has the effect of protecting the DNA from the related restriction endonuclease. However, there are methylases with minimal specificity. For example, Sss I methylase will methylate cytosine residues in the sequence 5' … CG … 3'. In this case, the methylated DNA will be protected from a wide variety of restriction endonucleases.
Some restriction endonucleases will only cut DNA at their recognition sites if the DNA is methylated (e.g. Dpn I).
Still other restriction endonucleases will cut both methylated and non-methylated DNA at their recognition sequences (e.g. BamH I).

dam and dcm methylation

DNA isolated from dam+dcm+ strains will not actually be cut by a modest subset of available restriction endonucleases:

Recognition sequence
Restriction enzyme
GATC
GmeATC
TGATCA
Bcl I
+
-
GATC
Mbo I
+
-
ATCGAT
Cla I
+
-
TCTAGA
Xba I
+
-
TCGA
Taq I
+
-
GAAGA
Mbo II
+
-
GGTGA
Hph I
+
-

DNA may have to be prepared from E. coli strains which are dam-dcm- in order to be cut by these enzymes.


DNA Polymerases

A wide variety of polymerases have been characterized and are commercially available. All DNA polymerases share two general characteristics:

  1. They add nucleotides to the 3'-OH end of a primer
  2. The order of the nucleotides in the nascent polynucleotide is template directed


In addition to the 5'->3' polymerase activity, polymerases can contain exonuclease activity. This exonuclease activity can proceed either in the 5'->3'direction, or in the 3'->5' direction.
Different polymerases have differing error rates of misincorporation, and different rates of polymerization.

E. coli DNA polymerase I
E. coli DNA polymerase I - Klenow Fragment
T4 DNA polymerase
T7 DNA polymerase
Taq DNA polymerase
M-MuLV Reverse Transcriptase
5'->3' exonuclease activity
*
*
3'->5' exonuclease activity
*
*
*
*
Error Rate (x10-6)
9
40
<1
15
285
Strand Displacement
*
Heat Inactivation
*
*
*
*
*



Uses of polymerases

The various activities of the different polymerases lend them to a variety of applications.

For example, restriction endonucleases can yield fragments of DNA with either 3' or 5' nucleotide "overhangs".


"Nick-translation"

This method is used to obtain highly radiolabeled single strand DNA fragments, which makes use of 5'->3' exonuclease activity present in some polymerases (E. coli DNA polymerase I, for example).





Nucleases

Nuclease BAL-31


Exonuclease III

Mung Bean Nuclease (isolated from mung bean sprouts)

Deoxyribonuclease I (DNAse I) from Bovine pancrease


Ligases

The following are different types of ligases and their characteristics.

T4 DNA ligase

Taq DNA ligase


T4 RNA ligase

T4 RNA ligase can be used for a variety of purposes including constructing RNA/DNA hybrid molecules.


DNA ligase (E. coli)


T4 polynucleotide kinase

T4 polynucleotide kinase can be used to phosphorylate the 5' end of such polynucleotides:




Calf intestinal phosphatase (CIP)



1998 Dr. Michael Blaber