<p><b>A</b>, thiamine-controlled cell growth of the shut-off strain (nmt-rad4) in which the endogenous promoter of <i>rad4<sup>+</sup></i> was replaced with a thiamine repressive <i>nmt81</i> promoter. Logarithmically growing cells were diluted in fivefold steps and spotted on EMM6S plates with (+) or without (-) thiamine. The plates were incubated at 30°C for 3 days before being photographed. <b>B</b>, thiamine-regulated expression of Rad4 was examined by Western blotting. Untagged Rad4, Rad4 with the deletion of whole C-terminus (ΔC) and HA-tagged Rad4 were expressed on a vector in the shut-off strain. Expression of Rad4 in the presence (+) or absence (-) of thiamine was examined by using anti-Rad4 antibodies (Top). The same membrane was stripped and blotted with anti-HA antibody (bottom). Asterisks indicate the cross-reacting materials. <b>C</b>, molecular architecture of Rad4 with relative locations of the newly isolated (solid circles) and previously reported (open circles) mutations and the AAD domain. The four BRCT repeats are marked by roman numerals. The regions covered by mutational PCRs in the genetic screening are also shown. <b>D</b>, sensitivities of the <i>rad4</i> mutants to HU and MMS were assessed by spot assay in the shut-off strain. The newly screened (top part) and the previously reported (lower part) mutations are marked on the right. Wild type cells, Δ<i>rad3</i> mutant, and the shut-off strain carrying an empty vector or the vector expressing wild-type Rad4 were used as controls.</p
