T7 endonuclease is an enzyme used as a molecular tool in biology. It helps scientists understand and manipulate DNA. This enzyme helps researchers investigate how DNA is structured and how changes within it can impact biological processes. It allows for precise analysis of DNA modifications in genetic studies.
What is T7 Endonuclease?
T7 endonuclease, commonly known as T7 Endonuclease I, is an enzyme originating from the T7 bacteriophage, a virus that infects Escherichia coli bacteria. This enzyme is encoded by gene 3 of the bacteriophage T7 genome and exists as a stable dimer. As a type of nuclease, it specifically functions as an endonuclease, meaning it cuts DNA strands from within, rather than acting on the ends.
In its natural context within the T7 virus life cycle, T7 endonuclease I is involved in processes such as DNA replication and recombination. It also contributes to the degradation of the host cell’s DNA, a common strategy employed by viruses to redirect cellular resources for their own propagation. This enzyme requires metal ions, such as magnesium, for its activity.
How T7 Endonuclease Functions
T7 endonuclease I operates by specifically targeting and cleaving DNA at sites where the double-stranded DNA structure is imperfect. It identifies regions containing mismatches, bulges, or other distortions. These structural anomalies in the DNA helix serve as its recognition signals.
The enzyme then proceeds to cut the DNA strand, typically at the first, second, or third phosphodiester bond located just before the detected mismatch. This action results in either a nick, which is a single-strand break, or a double-strand break, depending on the nature of the recognized imperfection. The enzyme is considered structure-selective, meaning its activity is primarily dictated by the physical shape of the DNA rather than a specific sequence of bases. Maintaining optimal conditions, such as temperatures below 42°C, is important, as higher temperatures can lead to less specific cutting activity.
Primary Applications in Genetic Research
T7 endonuclease I is widely utilized in genetic research, particularly for validating gene-editing experiments. Its primary application involves detecting mutations or insertions/deletions, often referred to as indels, introduced by gene-editing tools like CRISPR-Cas9, TALENs, and Zinc-finger nucleases. When these gene-editing systems make changes to DNA, the modified DNA strands, if mixed with original unmodified strands, can form mismatched regions.
A common method employing this enzyme is the T7 Endonuclease I (T7E1) assay. In this assay, genomic DNA is first extracted from the cells that have undergone gene editing. The specific region of interest, which includes the potential gene-editing site, is then amplified using a polymerase chain reaction (PCR). A high-fidelity DNA polymerase is selected for this step to prevent new mutations from being introduced during the amplification process.
After amplification, the PCR products are denatured by heating, separating the double-stranded DNA into single strands. These single strands are then allowed to reanneal by slowly cooling the mixture. If gene editing has successfully introduced an indel, some of the modified DNA strands will pair with unmodified, wild-type strands, forming heteroduplexes that contain mismatches or small loops. The T7 endonuclease I enzyme is then added to this reannealed DNA mixture.
The enzyme specifically cleaves these heteroduplex DNA molecules at the sites of mismatches. The resulting DNA fragments, which are shorter than the original PCR product, are then separated and visualized using gel electrophoresis. By comparing the intensity of the cleaved bands to the uncut bands, researchers can estimate the efficiency of the gene-editing event within a cell population. The T7E1 assay is a cost-effective and relatively simple technique for quickly assessing gene-editing outcomes.