CHARACTERIZATION OF 2 NOVEL SINGLE-STRANDED DNA-SPECIFIC AUTONOMOUSLYREPLICATING SEQUENCE-BINDING PROTEINS FROM SACCHAROMYCES-CEREVISIAE, ONE OF WHICH IS ADENYLOSUCCINATE SYNTHETASE

We report here the identification and characterization of two novel pr oteins from Saccharomyces cerevisiae that bind to the T-rich strand of the core consensus autonomously replicating sequence (ARS) in a highl y specific manner. The two proteins, 40 and 45 kDa in size, can be dis tinguished by multiple criteria from each other and from the 65-kDa ss ARS-T-binding protein identified recently in our laboratory (Schmidt, A. M. A., Herterich, S. U., and Krauss, G. (1991) EMBO J. 10, 981-985) . The specificity of binding is inferred from gel shift and nuclease-f ootprinting experiments using single-stranded probes containing the co re consensus ARS. With a 321-nucleotide single-stranded ARS1 fragment, specific protection of the A and B1 domain against DNase I digestion is observed. Partial amino acid sequencing and enzymatic assays identi fy the 45-kDa protein as adenylosuccinate synthetase, an enzyme necess ary for the de novo synthesis of adenylate.