Purification and Characterization of SMARCAL1 Orthologs in an E. coli Expression System

• Author / Creator

  Burkat, Michael

• SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) is a SWI2/SNF2 chromatin remodeler. It reanneals stalled replication forks to maintain genome integrity. It contains HARP domains that are necessary for the annealing activity. This thesis pursues structural studies of SMARCAL1 to understand its molecular mechanism. We establish an efficient E. coli expression system for the production of R. norvegicus and C. elegans SMARCAL1 orthologs. High yield and purity of six R. norvegicus proteins was obtained. One full length and five truncation mutants were designed to conserve the helicase domain and at least one HARP. These purified proteins were characterized and compared to an insect cell expressed human SMARCAL1 protein. DNA binding was assessed and the purified proteins displayed the typical preferential binding to fork DNA substrates. However, we determined that the DNA-stimulated ATPase activity reached similar catalytic rates for both fork and double stranded DNA.

• Subjects / Keywords

  • SWI2/SNF2
  • SMARCAL1
  • Crystallography

• Graduation date

  Fall 2013

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3C824P2C

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.