Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

Abstract

Work was conducted to find a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. A protein was isolated that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase .alpha. and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a MW of .apprx. 40,000. At apparent saturation there is 1 protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. There are 7500-30,000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. It is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.