The Werner syndrome gene product co-purifies with the DNA replication complex and interacts with PCNA and topoisomerase I.
Werner syndrome (WS) is a recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases. To understand the molecular basis of this disease, we deleted a segment of the murine Wrn gene and created Wrn-deficient embryonic stem (ES) cells. At the molecular level, wild type-but not mutant-WS protein co-purifies through a series of centrifugation, chromatography, and sucrose gradient steps with the well characterized 17 S multiprotein DNA replication complex. Furthermore, wild type WS protein co-immunoprecipitates with a prominent component of the multiprotein replication complex, proliferating cell nuclear antigen (PCNA). In vitro studies also indicate that PCNA binds to a region in the N terminus portion of the WS protein containing a potential 3'-5' exonuclease domain. Finally, human WS protein also co-immunoprecipitates with both PCNA and topoisomerase I. These results suggest that the WS protein interacts with several components of the DNA replication fork.
Lebel
M
M
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Spillare
E A
EA
Harris
C C
CC
Leder
P
P
D000818:Animals; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D006801:Humans; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D053484:RecQ Helicases; D018741:Replication Origin; D017384:Sequence Deletion; D014898:Werner Syndrome / Q000235:genetics*; D000071657:Werner Syndrome Helicase
D018809:Proliferating Cell Nuclear Antigen; D005090:Exodeoxyribonucleases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; D004264:DNA Topoisomerases, Type I