Use of monoclonal antibodies in the functional characterization of the Saccharomyces cerevisiae Sep1 protein.
The Saccharomyces cerevisiae strand-exchange protein 1 (Sep1 also known as Xrn1, Kem1, Rar5, Stp beta/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double-stranded and circular single-stranded DNA in vitro. To delineate the mechanism by which Sep1 acts in the strand-exchange reaction, we analyzed mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1 in vitro activity. Of 12 class-G immunoglobulins tested, four were found to consistently inhibit the Sep1-mediated strand-exchange reaction. The inhibiting antibodies were tested for inhibition of a variety of Sep1-catalyzed DNA reactions including exonuclease activity on double-stranded and single-stranded DNA, renaturation of complementary single-stranded DNA and condensation of DNA into large aggregates. All four inhibiting antibodies had no effect on the exonuclease activity of Sep1. Three antibodies specifically blocked DNA aggregation. In addition, one antibody inhibited renaturation of complementary single-stranded DNA. This inhibition pattern underlines the importance of condensation of DNA into large aggregates in conjunction with double-stranded DNA exonuclease activity for the in vitro homologous pairing activity of Sep1. The implications of these data for the interpretation of proteins which promote homologous pairing of DNA are discussed, in particular in light of the reannealing activity of the p53 human tumor-suppressor protein.
Holler
A
A
Institute of General Microbiology, University of Bern, Switzerland.
Bashkirov
V I
VI
Solinger
J A
JA
Reinhart
U
U
Heyer
W D
WD
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005092:Exonucleases / Q000378:metabolism; D005095:Exoribonucleases; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D003851:Deoxyribonucleases; D005092:Exonucleases; D005095:Exoribonucleases; C068398:XRN1 protein, S cerevisiae