Nonadditivity in the Recognition of Single-Stranded DNA by the Schizosaccharomyces pombe Protection of Telomeres 1 DNA-Binding Domain, Pot1-DBD
- 11 June 2009
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 48 (29), 6864-6875
- https://doi.org/10.1021/bi900307x
Abstract
The Schizosaccharomyces pombe protection of telomeres 1 (SpPot1) protein recognizes the 3′ single-stranded ends of telomeres and provides essential protective and regulatory functions. The ssDNA-binding activity of SpPot1 is conferred by its ssDNA-binding domain, Pot1-DBD (residues 1−389), which can be further separated into two distinct domains, Pot1pN (residues 1−187) and Pot1pC (residues 188−389). Here we show that Pot1pC, like Pot1pN, can function independently of Pot1-DBD and binds specifically to a minimal nonameric oligonucleotide, d(GGTTACGGT), with a KD of 400 ± 70 nM (specifically recognized nucleotides in bold). NMR chemical shift perturbation analysis indicates that the overall structures of the isolated Pot1pN and Pot1pC domains remain intact in Pot1-DBD. Furthermore, alanine scanning reveals modest differences in the ssDNA-binding contacts provided by isolated Pot1pN and within Pot1-DBD. Although the global character of both Pot1pN and Pot1pC is maintained in Pot1-DBD, chemical shift perturbation analysis highlights localized structural differences within the G1/G2 and T3/T4 binding pockets of Pot1pN in Pot1-DBD, which correlate with its distinct ssDNA-binding activity. Furthermore, we find evidence for a putative interdomain interface on Pot1pN that mediates interactions with Pot1pC that ultimately result in the altered ssDNA-binding activity of Pot1-DBD. Together, these data provide insight into the mechanisms underlying the activity and regulation of SpPot1 at the telomere.Keywords
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