Analyses of the interaction of WRNIP1 with Werner syndrome protein (WRN) in vitro and in the cell.
Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.
Kawabe
Yoh-ichi
Y
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Sendai 980-8578, Japan
Seki
Masayuki
M
Yoshimura
Akari
A
Nishino
Katsuaki
K
Hayashi
Tomoko
T
Takeuchi
Takashi
T
Iguchi
Sohta
S
Kusa
Yumiko
Y
Ohtsuki
Makoto
M
Tsuyama
Takashi
T
Imamura
Osamu
O
Matsumoto
Takehisa
T
Furuichi
Yasuhiro
Y
Tada
Shusuke
S
Enomoto
Takemi
T
D000074183:ATPases Associated with Diverse Cellular Activities; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D002645:Chickens; D004265:DNA Helicases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012854:Sister Chromatid Exchange; D020798:Two-Hybrid System Techniques; D014898:Werner Syndrome / Q000235:genetics / Q000378:metabolism; D000071657:Werner Syndrome Helicase
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; C430237:WRNIP1 protein, human; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; C499253:Wrn protein, mouse