Affinity chromatography on double-stranded (ds) and single-stranded (ss) DNA-cellulose columns was employed to find analogs of the Escherichia coli and T4 single-stranded DNA binding proteins (SSB proteins) in calf thymus. The interaction of several purified SSB proteins with the pure DNA-polymerase-α-primase complex on DNA synthesis on activated DNA and on primase-initiated M13 DNA served as a criterion for a possible involvement of one of these proteins in the process of DNA replication. Two SSB proteins were purified to essential homogeneity. These most abundant proteins exhibited apparent relative molecular masses of 35000 (SSB-35) and 37000 (SSB-37) for the protomers and 140000 and 80000 for the native enzymes. Both proteins resisted elution with 0.5 mg/ml dextran sulfate and were eluted from the ssDNA-cellulose with 0.2 M and 1 M NaCl, respectively. SSB-35 stimulated the DNA-polymerase-α–primase complex from the same organism up to fivefold over a broad range of DNA covering. By contrast, SSB-37 inhibited the primase-initiated replication of M13 DNA. Like most eukaryotic SSB proteins, these proteins showed a 300-fold preference for binding to ssDNA over dsDNA in a nitrocellulose filter binding assay, as well as strong binding to several DNA and RNA homopolymers. Furthermore, we provide evidence for a cooperative mode of binding for SSB-37. Although SSB-35 and SSB-37 behave as typically eukaryotic SSB proteins in all assays employed, we tested these SSB proteins for dehydrogenase activities as well. SSB-35 was found to be identical with lactate dehydrogenase and SSB-37 was identical with a dimeric form of glyceraldehyde-3-phosphate dehydrogenase. These results imply that further studies are mandatory in order to prove the authenticity of eukaryotic SSB proteins.
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- SSB protein
single-stranded DNA-binding protein
unwinding protein 1
sodium dodecyl sulfate
polyacrylamide gel electrophoresis
Lactate dehydrogenase (EC 188.8.131.52)
glyceraldehyde-3-phosphate-dehydrogenase (EC 184.108.40.206)
DNA polymerase (EC 220.127.116.11)
malate dehydrogenase (EC 18.104.22.168)
catalase (EC 22.214.171.124)
polynucleotide kinase (EC 126.96.36.199)
carbanonic anhydrase (EC 188.8.131.52)
lysozyme (EC 184.108.40.206)