FIG. 2.
Nbs1 associates with E2F1. (A) The N terminus of Nbs1 associates with E2F1. The N terminus of Nbs1 (p95N; aa 1 to 180) was tested for association with E2F1 (aa 284 to 416), empty vector, or a nonspecific control (SNF1) by yeast two-hybrid testing. Positive (Nbs1) and negative (vector and SNF4) controls for E2F1 association are shown. Growth on Trp-Leu-Ade and the Trp-Leu control plates is shown; results on Trp-Leu-His plates were identical. (B) Coimmunoprecipitation of Nbs1 with E2F1 in wild-type but not NBS cells. Lysates were prepared from wild-type (721) or NBS (DST) lymphoblasts, and immunoprecipitations (IP) were performed with E2F1 or E2F2 antibodies or with Nbs1 or preimmune (PI) antiserum. The immune complexes were resolved by SDS-PAGE and serially immunoblotted (IB) with Nbs1 antiserum, E2F1, E2F2, and Rb antibodies. Reciprocal IPs from murine embyronic stem (ES) cell extracts were carried out with Nbs1 antiserum 93, E2F1 antiserum 70, and Mre11 antiserum 59 (58) immunoblotted with Nbs1 93 and KH95. (C) Nbs1p70 associates with Mre11 but not E2F1. GAL4 DNA binding domain fusions of full-length Nbs1 or Nbs1p70 were tested for interaction with GAL4 activation domain fusions of E2F1 (aa 284 to 416), Mre11, empty vector, or a nonspecific control (SNF4). Growth on Trp-Leu-His and the Trp-Leu control plates is shown; results on Trp-Leu-Ade plates were identical. (D) Deletion analysis of the E2F1-Nbs1 interaction domain. Various fragments of E2F1 were cloned as fusions to the GAL4 activation domain and were tested for the ability to associate with full-length Nbs1 fused to the GAL4 DNA binding domain. The portions of each E2F1 fusion protein are shown as bars under the diagram depicting the locations of known motifs and interaction domains in E2F1 (55). A positive interaction (+) was defined as growth on both His and Ade test plates, and a weak interaction (+/−) was scored as growth on His plates only. The shaded boxes represent minimally defined regions of E2F1 necessary for Nbs1 association.