Single‐stranded DNA binding protein from calf thymus
- 1 December 1992
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 210 (3) , 855-865
- https://doi.org/10.1111/j.1432-1033.1992.tb17489.x
Abstract
A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA-cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius approximately 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11-kDa complex. The ssDNA binding protein (SSB) covered approximately 20-25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-alpha-primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA).(dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA).(dT)14; with poly(dT).(rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase alpha to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300-600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentration-dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.Keywords
This publication has 38 references indexed in Scilit:
- Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductasesPublished by Elsevier ,2003
- A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complexEuropean Journal of Biochemistry, 1989
- Immunoaffinity-purified DNA polymerase .alpha. displays novel propertiesBiochemistry, 1987
- Processivity of the DNA polymerase .alpha.-primase complex from calf thymusBiochemistry, 1987
- Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDaEuropean Journal of Biochemistry, 1986
- Salt-dependent changes in the DNA binding co-operativity of Escherichia coli single strand binding proteinJournal of Molecular Biology, 1986
- Analysis of secondary structures in M13mp8 (+) single‐stranded DNA by the pausing of DNA polymerase αEuropean Journal of Biochemistry, 1985
- Replication of M13mp7 single‐stranded DNA in vitro by the 9‐S DNA polymerase α from calf thymusEuropean Journal of Biochemistry, 1984
- Purification of a 9S DNA polymerase .alpha. species from calf thymusBiochemistry, 1981
- Rate of DNA replication fork movement within a single mammalian cellJournal of Molecular Biology, 1980