The Deinococcus radiodurans-encoded HU protein has two DNA-binding domains

Deinococcus radiodurans is known for its remarkable DNA repair capabilities, particularly after exposure to extreme radiation. This ability is thought to involve histone-like proteins such as HU, which are essential for DNA binding and structural stabilization. Prior research has shown that HU proteins generally bind DNA and influence recombination processes. However, the specific role of the N-terminal extension in D. radiodurans HU remains unclear. No prior work had resolved how this extension affects DNA binding specificity. This gap motivated the current study to investigate the functional role of the N-terminal domain in DrHU. Understanding how this domain modulates DNA interactions could clarify its role in DNA repair. The study focuses on how the N-terminal extension influences binding to different DNA structures. This work aims to distinguish between DNA binding modes in full-length versus truncated DrHU. The findings may help explain how DrHU contributes to genome stability in D. radiodurans.

This study aimed to determine how the N-terminal domain of DrHU influences DNA binding. The researchers hypothesized that this domain modulates DrHU’s interaction with DNA structures. They focused on comparing full-length DrHU with a truncated version lacking the N-terminal extension. Their goal was to assess binding differences to linear DNA, nicks, gaps, and four-way junctions. The study sought to clarify whether the N-terminal domain alters binding specificity. They also aimed to determine if the truncated DrHU could still bind DNA junctions. The researchers wanted to understand how the N-terminal extension affects DNA recognition. Their findings could explain how DrHU preferentially stabilizes DNA junctions in vivo.

The researchers used electrophoretic mobility shift assays to assess DNA binding by DrHU and deltaDrHU. They performed DNA footprinting to identify protected regions on DNA. The assays compared binding to linear DNA versus DNA with nicks or gaps. They also tested binding to four-way junction DNA structures. The team measured half-maximal saturation concentrations for DNA binding. They used Western blotting to confirm the presence of extended DrHU in vivo. The truncated DrHU was constructed by removing the N-terminal domain. The study compared binding affinities and site sizes between the two forms of DrHU.

The truncated DrHU binds DNA with a site size of approximately 11 bp, while full-length DrHU requires DNA longer than 50 bp. DeltaDrHU distinguishes between linear DNA and DNA with nicks or gaps. Full-length DrHU binds to junction arms, whereas deltaDrHU protects the junction crossover. DeltaDrHU binds four-way junction DNA with a half-maximal saturation of 1.4 nM. In contrast, full-length DrHU requires 20 nM for the same DNA. The N-terminal domain modulates DrHU’s binding mode to DNA structures. The truncated form preferentially binds four-way junctions. These findings suggest the N-terminal domain changes how DrHU interacts with DNA.

The N-terminal domain of DrHU significantly modulates DNA binding behavior. The truncated DrHU binds DNA with higher specificity to four-way junctions. Full-length DrHU requires longer DNA for stable binding. The N-terminal extension alters binding to junctions, nicks, and gaps. The truncated form protects the junction crossover rather than the arms. These findings suggest the N-terminal domain changes DrHU’s binding mode. The study supports a role for DrHU in stabilizing DNA junctions in vivo. The presence of extended DrHU in vivo confirms its functional relevance.