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A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11-389.

Abstract：

:The protection of telomeres 1 (Pot1) proteins specifically recognize the single-stranded 3' end of the telomere, an activity essential for sustained cellular viability and proliferation. The current model for the telomeric single-stranded DNA (ssDNA) binding activity of Schizosaccharomyces pombe Pot1 is based on a 20 kDa fragment, Pot1pN. Recent biochemical studies suggest that SpPot1 contains a larger ssDNA-binding domain and we have identified a novel ssDNA-binding domain similar in size to the human Pot1 domain. This domain, Pot1(1-389), binds extremely tightly to an oligonucleotide consisting of two conserved hexameric S. pombe telomere repeats, d(GGTTACGGTTAC), with an affinity approximately 4000-fold tighter than Pot1pN binds its cognate ssDNA. The Pot1(1-389)/ssDNA complex exhibits a half-life of 53 min, consistent with that estimated for full-length SpPot1 and significantly longer than that of Pot1pN. Single nucleotide substitutions reveal that, in contrast to Pot1pN, tandem trinucleotide repeats (GTT) within d(GGTTACGGTTAC) are specifically recognized by Pot1(1-389). Interestingly, certain single nucleotide substitutions that impacted Pot1pN binding exhibited no effect on binding affinity by Pot1(1-389). However, these substitutions reduced binding affinity when simultaneously substituted in each hexameric repeat. The non-additive nature of these substitutions suggests that certain nucleotides are coupled through the ability of the flexible ssDNA oligonucleotide to adopt alternate, thermodynamically equivalent conformations. The biochemical behavior of Pot1(1-389) is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making Pot1(1-389) a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Croy JE,Podell ER,Wuttke DS

doi

10.1016/j.jmb.2006.06.002

subject

Has Abstract

pub_date

2006-08-04 00:00:00

pages

80-93

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(06)00693-0

journal_volume

361

pub_type

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A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11-389.