The Unusual Story of Endonuclease V
A remarkable family of molecular guardians with surprising dual functions across different organisms
In the bustling metropolis of a living cell, where genetic information is constantly under threat, a remarkable family of molecular guardians works tirelessly to maintain order. Among these protective proteins exists endonuclease V (EndoV), an enzyme full of surprises. Initially discovered as a bacterial DNA repair specialist, EndoV was thought to have a straightforward job: finding and removing damaged DNA bases before they could cause harmful mutations. But as scientists dug deeper, they uncovered a startling secret—this versatile enzyme also operates in the world of RNA, suggesting functions far beyond what was initially imagined 1 4 .
The story of Endonuclease V is one of scientific rediscovery and evolving understanding. For decades following its initial identification in 1977, researchers believed they knew what EndoV did. Then, twist after twist revealed that this enzyme operates differently across species, with distinct roles in prokaryotes versus eukaryotes, and possesses unexpected talents that have reshaped our understanding of cellular maintenance 1 .
This is the story of an unusual DNA repair enzyme that turned out to be much more than first meets the eye.
The Endonuclease V story begins in the laboratory of Stuart Linn at the University of California, Berkeley, where in 1977 it was identified as the "fifth endonuclease" in Escherichia coli (E. coli) 1 . Initially, researchers characterized it as an enzyme that could nick DNA at various damaged sites, including those caused by ultraviolet light or abnormal bases 1 . The corresponding gene was named nfi 1 .
Endonuclease V discovered as the "fifth endonuclease" in E. coli
Identified as deoxyinosine 3' endonuclease by Kow and Weiss
Crystal structure of T. maritima EndoV solved
Human EndoV discovered to be an inosine-specific ribonuclease
To understand EndoV's job, we must first appreciate the problem it solves: deamination, a common type of DNA damage. Deamination occurs when the amino groups (-NH₂) on DNA bases are removed and replaced with keto groups (=O), fundamentally changing their base-pairing properties 1 .
When adenine loses its amino group, it becomes hypoxanthine, which pairs with cytosine instead of thymine 1 2 . Similarly, cytosine deamination creates uracil, which pairs with adenine instead of guanine 1 . If left unrepaired, these changes lead to mutations during DNA replication, potentially contributing to diseases like cancer 2 .
| Original Base | Deaminated Product | Pairs With | Resulting Mutation |
|---|---|---|---|
| Adenine (A) | Hypoxanthine (Hx) | Cytosine (C) | A:T → G:C |
| Cytosine (C) | Uracil (U) | Adenine (A) | G:C → A:T |
| Guanine (G) | Xanthine (X) | - | Mutagenic/Blocks replication |
What makes EndoV unusual is its repair mechanism. Unlike many DNA repair enzymes that remove damaged bases, EndoV takes a different approach. It cleaves the DNA backbone specifically at the second phosphodiester bond 3' to the lesion 1 2 . This creates a nick with 3'-hydroxyl and 5'-phosphate ends, but leaves the damaged nucleotide in place 1 . This distinctive cleavage pattern—an offset nick one nucleotide downstream from the damage—is a signature feature of EndoV 2 .
This repair pathway, known as Alternative Excision Repair (AER), is biochemically distinct from the more familiar Base Excision Repair (BER) and Nucleotide Excision Repair (NER) pathways 9 . While BER involves removing the damaged base itself via glycosylase activity, and NER removes a segment containing the damage, AER simply nicks the DNA strand next to the lesion, requiring additional enzymes to complete the repair process 9 .
EndoV cleaves at the second phosphodiester bond 3' to the deaminated base (hypoxanthine)
The 2009 crystal structure of Thermotoga maritima EndoV bound to DNA revealed the molecular secrets behind its function 2 . Researchers discovered a remarkable structural feature: a PYIP wedge motif (named for its proline-tyrosine-isoleucine-proline amino acid sequence) that acts as a minor-groove damage sensor 2 .
The tyrosine residue (Tyr80) in this motif stacks against the base adjacent to the lesion and helps stabilize the distorted DNA structure 2 . Mutations in this tyrosine severely compromise EndoV's ability to bind and process damaged DNA, highlighting its importance 2 .
Meanwhile, the damaged base itself is rotated approximately 90 degrees into a specialized recognition pocket about 8Å from the catalytic site 2 . This recognition pocket is lined with hydrophobic residues that interact with the damaged base, providing specificity for the repair process 2 .
One of the most fascinating aspects of EndoV biology is the dramatic functional shift between prokaryotic and eukaryotic versions of the enzyme.
In bacteria like E. coli, EndoV functions primarily as a DNA repair enzyme with remarkably broad specificity 8 9 . While its main substrate appears to be deaminated adenine (hypoxanthine), it can also recognize and process:
This broad substrate range reflects EndoV's general ability to recognize helical distortions in DNA rather than specific chemical lesions 2 .
The big surprise came when researchers discovered that mammalian EndoV, including the human version, shows little activity on DNA but instead functions as an inosine-specific ribonuclease 4 5 . This represents a dramatic evolutionary shift in function.
Human EndoV:
This suggests that in higher organisms, EndoV has been repurposed for RNA metabolism rather than DNA repair . Since inosine in RNA is frequently introduced by specific editing enzymes (ADARs) as a normal regulatory mechanism, this implies EndoV might play a role in processing edited RNAs or quality control of RNA molecules 4 5 .
| Feature | Prokaryotic EndoV | Eukaryotic EndoV |
|---|---|---|
| Primary substrate | DNA | RNA |
| Cellular localization | Nuclear | Cytoplasmic/Nucleolar |
| Main function | DNA repair | RNA metabolism |
| Cofactor requirement | Mg²⁺ or Mn²⁺ | Mg²⁺ or Mn²⁺ |
| Cleavage site | 2nd phosphodiester bond 3' to lesion | 2nd phosphodiester bond 3' to inosine |
| Biological role | Genome maintenance | RNA processing/quality control? |
For years, scientists were puzzled by the human version of EndoV. The enzyme was highly conserved evolutionarily, yet recombinant human ENDOV showed little activity on inosine-containing DNA—supposedly its primary substrate 4 . This paradox continued until 2013, when researchers from Norway and Japan independently made a startling discovery: human EndoV is actually an inosine-specific ribonuclease 4 .
The critical clue came from cellular localization studies. When researchers fused human EndoV to green fluorescent protein, they observed that it wasn't located in the nucleus as expected for a DNA repair enzyme. Instead, it was found in the cytoplasm and nucleoli—compartments rich in RNA 4 . This spatial clue prompted scientists to question whether human EndoV might actually target RNA instead of DNA.
The experiments yielded clear and compelling results:
| Enzyme | ssRNA with inosine | dsRNA with inosine | ssDNA with inosine | dsDNA with inosine |
|---|---|---|---|---|
| E. coli EndoV | ++ | ++ | +++ | +++ |
| Human ENDOV | +++ | ++ | - | - |
| T. brucei EndoV | +++ | - | + | - |
Key: (+++) Strong activity, (++) Moderate activity, (+) Weak activity, (-) No detectable activity
Data compiled from 4 7
This discovery fundamentally changed our understanding of EndoV's biological role and evolutionary conservation. It revealed that:
The findings opened new research directions into EndoV's potential roles in RNA quality control and the metabolism of inosine-containing RNAs produced by editing enzymes 4 5 .
Studying an enzyme as versatile as EndoV requires specialized reagents and tools. Here are some essential components of the EndoV research toolkit:
| Reagent/Tool | Function/Application | Examples/Specifics |
|---|---|---|
| Recombinant EndoV proteins | Biochemical characterization of enzyme activity | E. coli EndoV, human ENDOV, T. maritima EndoV 4 8 |
| Oligonucleotide substrates | Testing enzyme specificity and kinetics | DNA/RNA with inosine, abasic sites, mismatches 4 7 |
| Site-directed mutants | Identifying critical residues for catalysis | D35A (E. coli), D52A (human) catalytic mutants 4 5 |
| Metal cofactors | Essential for catalytic activity | Mg²⁺, Mn²⁺ 4 |
| Crystallization reagents | Structural studies of enzyme-substrate complexes | Used to solve T. maritima EndoV-DNA structures 2 |
The story of Endonuclease V continues to evolve. While we've made significant progress in understanding its molecular mechanisms and biochemical activities, many questions remain unanswered, particularly for the eukaryotic versions of the enzyme.
Recent research suggests that human EndoV's activity may be controlled by ATP levels and its recruitment to cytoplasmic stress granules under certain conditions 5 .
What is clear is that Endonuclease V represents a fascinating example of evolutionary adaptation. From its origins as a DNA repair enzyme in bacteria to its repurposing as an RNA-processing enzyme in humans, this unusual enzyme continues to surprise and delight scientists with its versatility and conservation across domains of life.
As research continues, Endonuclease V may well yield new insights into cellular maintenance mechanisms, the evolutionary repurposing of protein folds, and potentially even new biotechnology applications in mutation detection and DNA manipulation 1 . For an enzyme that was discovered over four decades ago, Endonuclease V continues to reveal new secrets about the intricate workings of the cell.