Skip to main content

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1.

Publication ,  Journal Article
Routh, ED; Creacy, SD; Beerbower, PE; Akman, SA; Vaughn, JP; Smaldino, PJ
Published in: J Vis Exp
March 18, 2017

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

March 18, 2017

Issue

121

Location

United States

Related Subject Headings

  • Telomere
  • RNA
  • Kinetics
  • Humans
  • Hela Cells
  • HeLa Cells
  • G-Quadruplexes
  • DNA Replication
  • DNA
  • DEAD-box RNA Helicases
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Routh, E. D., Creacy, S. D., Beerbower, P. E., Akman, S. A., Vaughn, J. P., & Smaldino, P. J. (2017). A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1. J Vis Exp, (121). https://doi.org/10.3791/55496
Routh, Eric D., Steven D. Creacy, Peter E. Beerbower, Steven A. Akman, James P. Vaughn, and Philip J. Smaldino. “A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1.J Vis Exp, no. 121 (March 18, 2017). https://doi.org/10.3791/55496.
Routh ED, Creacy SD, Beerbower PE, Akman SA, Vaughn JP, Smaldino PJ. A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1. J Vis Exp. 2017 Mar 18;(121).
Routh, Eric D., et al. “A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1.J Vis Exp, no. 121, Mar. 2017. Pubmed, doi:10.3791/55496.
Routh ED, Creacy SD, Beerbower PE, Akman SA, Vaughn JP, Smaldino PJ. A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1. J Vis Exp. 2017 Mar 18;(121).

Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

March 18, 2017

Issue

121

Location

United States

Related Subject Headings

  • Telomere
  • RNA
  • Kinetics
  • Humans
  • Hela Cells
  • HeLa Cells
  • G-Quadruplexes
  • DNA Replication
  • DNA
  • DEAD-box RNA Helicases