In vitro Evidence that Purified Yeast Rad27 and Dna2 are not Stably Associated with Each Other Suggests that an Additional Protein(s) is Required for a Complex Formation

The Saccharomyces cerevisiae Rad27, a structure-specific endonuclease for the Okazaki fragment maturation has been known to interact genetically and biochemically with Dna2, an essential enzyme for DNA replication. In an attempt to define the significance of the interaction between the two enzymes, we expressed and purified both Dna2 and Rad27 proteins. In this report, Rad27 could not form a complex with Dna2 in the three different analyses. The analyses included glycerol gradient sedimentation, protein-column chromatography, and coinfection of baculoviruses followed by affinity purification. This is in striking contrast to the previous results that used crude extracts. These results suggest that the interaction between the two proteins is not sufficiently stable or indirect, and thus requires an additional protein(s) in order for Rad27 and Dna2 to form a stable physical complex. This result is consistent with our genetic findings that Schizosaccharomyces pombe Dna2 is capable of interacting with several proteins that include two subunits of polymerase d, DNA ligase I, as well as Fen-1. In addition, we found that the N-terminal modification of Rad27 abolished its enzymatic activity. Thus, as suspected, we found that on the basis of the structure determination, N-terminal methionine indeed plays an important role in the nucleolytic cleavage reaction.
Publisher
한국생화학회
Issue Date
2000-03
Language
KOR
Citation

JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY - 등재, v.33, no.2, pp.155 - 161

ISSN
1225-8687
URI
http://hdl.handle.net/10203/74822
Appears in Collection
BS-Journal Papers(저널논문)
Files in This Item
There are no files associated with this item.
  • Hit : 168
  • Download : 0
  • Cited 0 times in thomson ci

qr_code

  • mendeley

    citeulike


rss_1.0 rss_2.0 atom_1.0