Abstract
A cloned cDNA corresponding to OsDMC1 from rice anther tissue was expressed in Escherichia coli. The OsDmc1 protein was largely present in the inclusion bodies of the cell lysatE., which was solubilized by 8.0 M urea containing buffeR., purified to homogeneity by Ni-CAM agarose column chromatography, followed by renaturation to its native state through stepwise dialysis against reduced concentrations of urea. The purified protein cross-reacted with anti-yeast Dmc1 antibodies. The binding efficiency observed with circular single-stranded DNA (ssDNA) was similar to that with circular double-stranded DNA (dsDNA). The binding to either DNA showed no ATP dependencE., but required 5–10 mM Mg2+ in the presence of ATP. Even though the protein binding to dsDNA was as efficient as it was to ssDNA, the former induced no DNA dependent ATPasE., whereas the binding to ssDNA stimulated a significant level of DNA dependent ATPase activity. OsDmc1–ssDNA complex, with its ATPase proficiency, also mediated renaturation of homologous complementary strands as well as assimilation of single strands into homologous supercoiled duplexes leading to D-loop formation. The D-loop formation was lowered by excess of OsDmc1 protein. This D-loop formation activity was promoted by non-hydrolyzable ATP analog, AMP-PNP and was not observed in absence of ATP or presence of ADP/ATP-γ-S. These properties reflected the classical hallmarks of a recombinase and represented the first biochemical characterization of a plant Dmc1 protein.
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Abbreviations
- ATP-:
-
γ-S: Adenosine 5′-O-(3-thio-triphosphate)
- BME:
-
β-mercaptoethanol
- BSA:
-
Bovine serum albumin
- DTT:
-
dithiothreitol
- Ni-CAM:
-
Nickel chelating affinity matrix
- PEI:
-
polyethyleneimine
- SS:
-
single-stranded
- DS:
-
double-stranded
- RF:
-
replicative form
- OsDmc1:
-
Oryza sativa disrupted meiotic cDNA1
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Kant, C.R., Rao, B.J. & Sainis, J.K. DNA binding and pairing activity of OsDmc1, a recombinase from rice. Plant Mol Biol 57, 1–11 (2005). https://doi.org/10.1007/s11103-004-5828-x
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DOI: https://doi.org/10.1007/s11103-004-5828-x