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Nakaoka, H, Nishiyama, A, Saito, M and Ishikawa, F (2012) Xenopus laevis Ctc1-Stn1-Ten1 (xCST) protein complex is involved in priming DNA synthesis on single-stranded DNA template in Xenopus egg extract. J. Biol. Chem. 287:619-27
The Ctc1-Stn1-Ten1 (CST) complex is an RPA (replication protein A)-like protein complex that binds to single-stranded (ss) DNA. It localizes at telomeres and is involved in telomere end protection in mammals and plants. It is also known to stimulate DNA polymerase α-primase in vitro. However, it is not known how CST accomplishes these functions in vivo. Here, we report the identification and characterization of Xenopus laevis CST complex (xCST). xCST showed ssDNA binding activity with moderate preference for G (guanine)-rich sequences. xStn1-immunodepleted Xenopus egg extracts supported chromosomal DNA replication in in vitro reconstituted sperm nuclei, suggesting that xCST is not a general replication factor. However, the immunodepletion or neutralization of xStn1 compromised DNA synthesis on ssDNA template. Because primed ssDNA template was replicated in xStn1-immunodepleted extracts as efficiently as in control ones, we conclude that xCST is involved in the priming step on ssDNA template. These results are consistent with the current model that CST is involved in telomeric C-strand synthesis through the regulation of DNA polymerase α-primase.
Animals; Base Sequence; Cell Nucleus/genetics; Cell Nucleus/metabolism; Chromatin/genetics; Chromatin/metabolism; Cloning, Molecular; DNA Replication; DNA, Single-Stranded/biosynthesis; DNA, Single-Stranded/genetics; DNA, Single-Stranded/metabolism; GC Rich Sequence/genetics; Humans; Male; Ovum/metabolism; Protein Transport; Spermatozoa/cytology; Spermatozoa/metabolism; Substrate Specificity; Telomere/genetics; Telomere/metabolism; Xenopus Proteins/genetics; Xenopus Proteins/metabolism; Xenopus laevis/genetics; Xenopus laevis/metabolism
|Gene product||Qualifier||GO Term||Evidence Code||with/from||Aspect||Extension||Notes||Status|
|GO:0070188: Stn1-Ten1 complex||
Figure 2 - assay shows that protein complex binds to ssDNA Figure 5 - assay shows that xStn1 immunodepletion does not affect DNA polymerase α-primase binding to ssDNA
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