Recombinant Tropheryma whipplei Holliday junction ATP-dependent DNA helicase RuvA (ruvA)
Description
Introduction
The ruvA gene encodes a DNA helicase that recognizes Holliday junctions and promotes branch migration, playing a vital role in genetic recombination and DNA repair . Specifically, RuvA is a component of the RuvABC complex, essential for resolving Holliday junctions formed during homologous recombination in bacteria .
Tropheryma whipplei
Tropheryma whipplei is a bacterium that causes Whipple's disease, a rare systemic illness affecting multiple organs in the body . Identification of T. whippelii relies on the amplification of its 16S rRNA gene, which is used as a standard diagnostic method . Molecular characterization using PCR-based techniques has improved the diagnosis of Whipple’s disease .
Holliday Junction and RuvA Function
Holliday junctions are four-way DNA structures that form during DNA recombination, replication, and repair . RuvA protein binds with high affinity to Holliday junctions . The Escherichia coli RuvA protein binds specifically to the Holliday structure, forming a complex with the RuvB motor protein, which facilitates the migration of the Holliday junction using ATP hydrolysis energy, expanding the heteroduplex region .
RuvA and RuvB Interaction
RuvA interacts directly with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along the DNA . This process, known as branch migration, is important for the formation of heteroduplex DNA . RuvA and RuvB proteins promote the unwinding of partially duplex DNA, with RuvA providing specificity by binding to the Holliday junction .
ATP Dependence
The RuvAB-mediated branch migration requires ATP to bypass UV-induced DNA lesions . The RuvB ATPase provides the motor force for branch migration . The efficiency of RuvA and RuvB is inversely related to the length of the duplex DNA .
RuvA Structure and Mechanism
RuvA proteins target the Holliday junction and facilitate the loading of RuvB helicase and RuvC endonuclease to form complexes that catalyze junction branch migration (RuvAB) and resolution (RuvABC) . The structure of the RuvA complex consists of either one or two RuvA tetramers, with charged grooves that channel incoming DNA . The 'acidic pins' in the center of the tetramer separate the DNA duplexes .
Synergistic Effect of ATP for RuvA–RuvB–Holliday Junction DNA Complex Formation
| Condition | % of Holliday junction DNA interacting with RuvB | % of Holliday junction DNA with one RuvB | % of Holliday junction DNA with two RuvBs | % of Holliday junction DNA with three RuvBs | % of Holliday junction DNA with four RuvBs | % of Holliday junction DNA with five RuvBs | % of Holliday junction DNA with six RuvBs |
|---|---|---|---|---|---|---|---|
| No nucleotide | 77 | 37 | 40 | N/A | N/A | N/A | N/A |
| ADP | 90 | N/A | N/A | 30 | 29 | N/A | N/A |
| ATPγS | 92 | N/A | N/A | N/A | 31 | 12 | N/A |
| ADP and ATPγS | 98 | N/A | N/A | 31 | 37 | 14 | 3 |
Product Specs
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Target Background
The RuvA-RuvB complex, in the presence of ATP, renatures cruciform structures in supercoiled DNA with palindromic sequences, suggesting a role in promoting strand exchange reactions during homologous recombination. RuvAB functions as a helicase, mediating Holliday junction migration through localized denaturation and reannealing. RuvA enhances the weak ATPase activity of RuvB in the presence of DNA.
KEGG: twh:TWT_268
STRING: 203267.TWT268
