Purification, Crystallization and Preliminary X-Ray Diffraction Analysis of Exodeoxyribonuclease III from Crenarchaeon &amp;lt;i&amp;gt;Sulfolobus tokodaii&amp;lt;/i&amp;gt; Strain 7

Shuichi Miyamoto,Chieko Naoe,Masaru Tsunoda,Kazuo T Nakamura

doi:10.4236/csta.2013.24021

Abstract

Exodeoxyribonuclease III (EXOIII) acts as a 3’→5’ exonuclease and is homologous to purinic/apyrimidinic (AP) endonuclease (APE), which plays an important role in the base excision repair pathway. To structurally investigate the reaction and substrate recognition mechanisms of EXOIII, a crystallographic study of EXOIII from Sulfolobus tokodaii strain 7 was carried out. The purified enzyme was crystallized by using the hanging-drop vapor-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 154.2, b = 47.7, c = 92.4 ?, β = 125.8° and diffracted to 1.5 ? resolution.

Highlights

A variety of mechanisms exist to repair damaged DNA and maintain a high degree of genomic stability within cells
AP sites are generated by spontaneous base loss [1], direct action of reactive oxygen species [2] and damage-specific DNA glycosylases as repair intermediates in the DNA base excision repair (BER) pathway [3,4]
AP sites are corrected by the BER pathway: AP endonucleases (APE) first recognize the AP site and cleave the phosphodiester backbone immediately 5’ of the AP site, leaving a free 3’-hydroxyl nucleotide end and a deoxyribose 5’-phospate as termini [8,9]

Summary

Introduction

A variety of mechanisms exist to repair damaged DNA and maintain a high degree of genomic stability within cells. Endonucleases specific for abasic or apurinic/ apyrimidinic (AP) sites are a major component of the cellular DNA repair machinery. AP sites are generated by spontaneous base loss [1], direct action of reactive oxygen species [2] and damage-specific DNA glycosylases as repair intermediates in the DNA base excision repair (BER) pathway [3,4]. The three dimensional structures of EXOIII from Escherichia coli (E. coli) [12] and human APE [13] have been elucidated, but structures of these two enzymes from the same species have yet to be determined. To reveal the structural basis for the reaction and substrate recognition mechanisms of these enzymes from the same organism, we crystallized EXOIII and APE from the crenarchaeon, Sulfolobus tokodaii strain 7 [14]. The enzyme consists of 241 amino acids (NCBI reference code NP_377894; UniProt ID F9VNP2), and has a molecular weight of 28.08 kDa and an isoelectric point of 8.72

Expression and Purification

Crystallization

Results and Discussion

Conclusion