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Restriction Enzymes

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...

Nucleotide Excision Repair

Nucleotide Excision Repair

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Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...

Nucleotide Excision Repair

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DNA Topoisomerases

DNA Topoisomerases

Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
Types and Mechanism of action
Topoisomerases are divided into two main types. Type I...

Base Excision Repair

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...

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Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

Published on: July 5, 2024

Endonucleases.

Nicole M Nichols¹

¹New England Biolabs, Inc., Ipswich, MA, USA.

Current Protocols in Molecular Biology

|January 13, 2011

Summary

This summary is machine-generated.

This unit details reaction conditions and applications for various endonucleases, including BAL 31, S1, mung bean, micrococcal nuclease, and DNase I. It also covers generating nonspecific breaks in double-stranded DNA (dsDNA).

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Area of Science:

Molecular Biology
Enzymology

Background:

Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains.
Understanding endonuclease activity is crucial for various molecular biology techniques.

Purpose of the Study:

To provide detailed reaction conditions for specific endonucleases.
To discuss potential applications of these enzymes.
To explain the generation of nonspecific breaks in double-stranded DNA (dsDNA).

Main Methods:

Detailed protocols for enzyme reactions.
Discussion of enzyme characteristics and applications.
Explanation of dsDNA cleavage mechanisms.

Main Results:

Specific reaction parameters for BAL 31 nuclease, S1 nuclease, mung bean nuclease, micrococcal nuclease, and DNase I are presented.
Guidelines for utilizing these enzymes in molecular biology are provided.
The principle of using endonucleases for nonspecific dsDNA breaks is elucidated.

Conclusions:

This unit serves as a practical guide to using a range of endonucleases.
The information facilitates the application of these enzymes in research and biotechnology.
Knowledge of endonuclease activity is essential for DNA manipulation and analysis.