Binding to the Minor Groove of the Double-Strand, Tau Protein Prevents DNA from Damage by Peroxidation

Yan Wei,Xing-Sheng Wang,Dong-Liang Wang,Ying Liu,Mei-Hua Qu,Qian Hua,Lan Chen,Rong-Qiao He

doi:10.1371/journal.pone.0002600

Abstract

Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H2O2/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical.

Highlights

Tau is a major microtubule-binding protein that is important for the assembly and stabilization of microtubules [1]
To investigate the minimum polynucleotide that is bound to tau protein, we synthesized a series of polynucleotides (11–26 bp) and incubated them with tau protein for electrophoretic mobility shifting assay (EMSA) (Table 1)
To investigate further whether the protein discriminated between 13 bp and other lengths of polynucleotides, 12 bp and 13 bp polynucleotides were incubated with tau and subjected to EMSA

Summary

Introduction

Tau is a major microtubule-binding protein that is important for the assembly and stabilization of microtubules [1]. The six isoforms of tau have been observed to bind to nucleolar organizer regions of acrocentric chromosomes and the fibrillar region of the nucleoli (the site for rRNA transcription) in some non-neuronal cells such as lymphocytes [5]. As shown by an in vitro assay (37uC), tau protein represses DNA replication, but does not affect RNA transcription in the presence of T7 RNA polymerase [11], behavior that is similar to histone.

Methods

Results

Conclusion