title: Drosophila Rrp1 domain structure as defined by limited proteolysis and biophysical analyses.
abstract: Drosophila Rrp1 is a DNA repair nuclease whose C-terminal region shares extensive homology with Escherichia coli exonuclease III, has nuclease activity, and provides resistance to oxidative and alkylating agents in repair-deficient E. coli strains. The N-terminal 421 amino acid region of Rrp1, which binds and renatures homologous single-stranded DNA, does not share homology with any known protein. Proteolysis by endoproteinase Glu-C (protease V8) reduces the Rrp1 protein to a single, cleavage-resistant peptide. The peptide (referred to as Rrp1-C274) begins with the sequence TKTTV, corresponding to cleavage between Glu-405 and Thr-406 of Rrp1. We determined that nuclease activity is intrinsic to Rrp1-C274 although altered when compared with Rrp1; 3'-exonuclease activity is reduced 210-fold, 3'-phosphodiesterase activity is reduced 6.8-fold, and no difference in apurinic/apyrimidinic endonuclease activity is observed. Rrp1 and Rrp1-C274 are both monomers with frictional coefficients of 2.2 and 1.4, respectively. Circular dichroism results indicate that Rrp1-C274 is predominantly alpha-helical, while the N-terminal 399 amino acids is predominantly random coil. These results suggest that Rrp1 may have a bipartite structural organization; a highly organized, globular C-terminal domain; and an asymmetric, protease-sensitive random coil-enriched N-terminal region. A shape model for this bipartite structure is proposed and discussed.
authors.affiliation: Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. reardon@niehs.nih.gov
authors.forename: B J
authors.initials: BJ
authors.lastname: Reardon
authors.forename: C R
authors.initials: CR
authors.lastname: Lombardo
authors.forename: M
authors.initials: M
authors.lastname: Sander
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D002942:Circular Dichroism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004330:Drosophila / Q000201:enzymology*; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000737:chemistry; D005092:Exonucleases / Q000378:metabolism; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D009713:Nucleotidyltransferases; C061900:Rrp1 protein, Drosophila; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C020133:exodeoxyribonuclease III; C022650:spleen exonuclease; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D010727:Phosphoric Diester Hydrolases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase