Hughes P, Baldacci G
CNRS-IFC1, Institut de Recherches sur le Cancer, UPR 9044, 7 rue Guy Moquet, BP 8, 94801 Villejuif Cedex, France.
Nucleic Acids Res. 1997 Oct 1;25(19):3881-8. doi: 10.1093/nar/25.19.3881.
In an effort to identify cellular helicases that mimic the action of SV40 large T-antigen, we performed replication protein A (RPA) affinity chromatography on cell extracts from the mouse mammary carcinoma cell line FM3A. In this way, a novel DNA helicase was isolated and purified to near homogeneity. The most purified fractions showed the presence of two proteins of 28 and 21 kDa. Both proteins interacted with 32P-labeled partially duplex DNA when bound to nitrocellulose membranes and were efficiently UV crosslinked to [alpha-32P]dATP. Helicase activity was strongly stimulated by RPA on DNA substrates containing duplex regions longer than 18 bp. Only weak stimulation was observed in the presence of Escherichia coli single strand DNA binding protein (SSB). The enzyme unwinds DNA in the 5'-3' direction in relation to the strand to which it binds. Only ATP and dATP were efficient as nucleoside triphosphate co-factors, and showed similar Km values of approximately 0.6 mM. The properties of this enzyme suggest that it may take part in reactions mediated by RPA such as those predicted to occur at replication forks or alternatively may function during DNA repair or recombination.
为了鉴定模拟SV40大T抗原作用的细胞解旋酶,我们对小鼠乳腺癌细胞系FM3A的细胞提取物进行了复制蛋白A(RPA)亲和层析。通过这种方法,分离并纯化了一种新型DNA解旋酶,使其接近均一性。纯化程度最高的组分显示存在两种蛋白质,分子量分别为28 kDa和21 kDa。当与硝酸纤维素膜结合时,这两种蛋白质都能与32P标记的部分双链DNA相互作用,并能有效地与[α-32P]dATP进行紫外线交联。在含有长度超过18 bp双链区域的DNA底物上,RPA能强烈刺激解旋酶活性。在大肠杆菌单链DNA结合蛋白(SSB)存在的情况下,仅观察到微弱的刺激作用。该酶相对于其结合的链,以5'-3'方向解旋DNA。只有ATP和dATP作为核苷三磷酸辅因子是有效的,并且显示出相似的Km值,约为0.6 mM。这种酶的特性表明,它可能参与由RPA介导的反应,如预计在复制叉处发生的反应,或者可能在DNA修复或重组过程中发挥作用。