Related Experiment Video
Updated: Jun 10, 2026
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
The type II restriction endonuclease MvaI has dual specificity
1Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
Nucleic Acids Research
|August 10, 2010
Summary
MvaI restriction enzyme cleaves DNA at specific sites. It can also nick DNA at different sequences when cytosines are methylated, revealing a novel methylation-directed activity.
Area of Science:
- Molecular Biology
- Enzymology
- Epigenetics
Background:
- Restriction endonucleases are enzymes that cleave DNA at specific recognition sites.
- DNA methylation, particularly of cytosine bases, plays a crucial role in gene regulation and genome stability.
- MvaI is a known restriction enzyme with a specific recognition sequence.
Purpose of the Study:
- To investigate the activity of MvaI restriction endonuclease on methylated DNA sequences.
- To characterize the novel methylation-directed cleavage activity of MvaI.
- To compare the cleavage properties of MvaI on its canonical and methylation-dependent sites.
Main Methods:
- In vitro enzymatic assays using synthetic DNA substrates with varying methylation patterns.
- Analysis of DNA cleavage products by gel electrophoresis.
- Characterization of MvaI activity on canonical (CCAGG/CCTGG) and related (CCGGG/CCCGG) sequences with C5-methylated cytosines.
Main Results:
- MvaI nicks the G-strand of a BcnI recognition site (CCGGG/CCCGG) when inner cytosines are C5-methylated.
- MvaI induces double-strand cleavage at overlapping, methylated BcnI sites within SmaI sites, generating blunt-ended fragments.
- Cleavage at the methylation-dependent site is less stringent and occurs at a lower rate compared to the canonical MvaI site.
Conclusions:
- MvaI exhibits dual activity: canonical cleavage and a novel methylation-directed cleavage on a different DNA sequence.
- This is the first reported instance of a restriction endonuclease possessing both standard and methylation-directed activities on distinct sequences.
- The findings expand our understanding of restriction enzyme mechanisms and their potential roles in epigenetic processes.
Related Concept Videos
01:11Restriction 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...
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
02:53CRISPR and crRNAs
Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
01:26DNA Bacteriophages
Bacteriophages, or phages, are viruses that specifically infect bacteria, utilizing their genetic material to hijack host cellular machinery for replication. DNA bacteriophages employ single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) genomes. These phages exhibit diverse replication strategies and host interactions, influencing their ecological roles and applications in biotechnology and medicine.ssDNA BacteriophagesssDNA phages, with their small genomes, utilize unique strategies to...
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
02:02DNA 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...
Types and Mechanism of action
Topoisomerases are divided into two main types. Type I...
01:28Inhibitors of Bacterial DNA Synthesis
Bacterial pathogens depend on precise and efficient DNA replication to sustain infection. Two type II topoisomerases—DNA gyrase and topoisomerase IV—are critical to this process, as they resolve DNA supercoiling and unlink chromosomes during replication. Fluoroquinolones, synthetic derivatives of quinolones, exploit this mechanism by stabilizing the transient DNA–enzyme cleavage complex, preventing strand religation, and causing lethal double-strand breaks. These antibiotics are selectively...