Figure 2.

Mrc1 has DNA binding activity. (A) A schematic view of Mrc1 and its mutant proteins. BP1 represents the basic patch 1 in the N-terminus of Mrc1 containing multiple basic residues, probably involved in DNA binding. Alanine and glutamine acid substitutions of three lysine residues (K323, K324, and K328) were designated as Mrc1-3KA and Mrc1–3KE, respectively. Mrc1-3D bears the phospho-mimetic mutations of three residues (T169, S215, and S229), which have been demonstrated to be phosphorylated by Hog1 in response to osmotic stress. (B) Silver staining of purified recombinant Mrc1 mutant proteins (3KA, 3KE, 3D) as well as WT. (C) Purified Mrc1 WT or 3KA mutant protein was incubated with ³²P-labelled ssDNA at the 5′-end. After incubation at 4°C for 30min, samples were analyzed via 5% native PAGE gels followed by autoradiography. E. coli SSB (single-stranded DNA binding protein) was used as a control.