Drosophila Rrp1 domain structure as defined by limited proteolysis and biophysical analyses

Drosophila Rrp1 is a DNA repair nuclease whose C-terminal region shares ext ensive homology with Escherichia coli exonuclease III, has nuclease activit y, and provides resistance to oxidative and alkylating agents in repair-def icient E. coli strains. The N-terminal 421 amino acid region of Rrp1, which binds and renatures homologous single-stranded DNA, does not share homolog y with any known protein. Proteolysis by endoproteinase Glu-C (protease V8) reduces the Rrp1 protein to a single, cleavage-resistant peptide. The pept ide (referred to as Rrp1-C274) begins with the sequence TKTTV, correspondin g to cleavage between Glu-405 and Thr-406 of Rrp1, We determined that nucle ase activity is intrinsic to Rrp1-C274 although altered when compared with Rrp1; 3'-exonuclease activity is reduced 210-fold, 3'-phosphodiesterase act ivity is reduced 6.8-fold, and no difference in apurinic/apyrimidinic endon uclease activity is observed. Rrp1 and Rrp1-C274 are both monomers with fri ctional coefficients of 2.2 and 1.4, respectively. Circular dichroism resul ts indicate that Rrp1-C274 is predominantly cu-helical, while the N-termina l 399 amino acids is predominantly random coil. These results suggest that Rrp1 may have a bipartite structural organization; a highly organized, glob ular C-terminal domain; and an asymmetric, protease-sensitive random coil-e nriched N-terminal region. A shape model for this bipartite structure is pr oposed and discussed.