Nucleic Acids Research, Vol 26, Issue 13 3165-3172, Copyright © 1998 by Oxford University Press
C Campbell and DP Romero
The RAD51 gene is a eukaryotic homolog of rec A, a critical component in
homologous recombination and DNA repair pathways in Escherichia coli . We
have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated
protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose
amino acid sequence is highly similar to representative Rad51 homologs from
other eukaryotic taxa. Recombinant Rad51 protein was purified to near
homogeneity following overproduction in a bacterial expression system. The
purified protein binds to both single- and double-stranded DNA, possesses a
DNA-dependent ATPase activity and promotes intermolecular ligation of
linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in
normally growing cells, treatment with UV light resulted in a >100-fold
increase in mRNA levels. This increase in mRNA was time dependent, but
relatively independent of UV dose over a range of 1400-5200 J/m2. Western
blot analysis confirmed that Rad51 protein levels increase upon UV
irradiation. Exposure to the alkylating agent methyl methane sulfonate also
resulted in substantially elevated Rad51 protein levels in treated cells,
with pronounced localization in the macronucleus. These data are consistent
with the hypothesis that ciliates such as T.thermophila utilize a
Rad51-dependent pathway to repair damaged DNA.
ARTICLES
Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila
Department of Pharmacology, Medical School, University of Minnesota, 3- 249 Millard Hall, 435 Delaware Street SE, Minneapolis, MN 55455, USA.
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