Skip to main content
Journal cover image

Structure-guided reprogramming of serine recombinase DNA sequence specificity.

Publication ,  Journal Article
Gaj, T; Mercer, AC; Gersbach, CA; Gordley, RM; Barbas, CF
Published in: Proceedings of the National Academy of Sciences of the United States of America
January 2011

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences > 10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with > 80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

Proceedings of the National Academy of Sciences of the United States of America

DOI

EISSN

1091-6490

ISSN

0027-8424

Publication Date

January 2011

Volume

108

Issue

2

Start / End Page

498 / 503

Related Subject Headings

  • Transposon Resolvases
  • Transgenes
  • Serine
  • Sequence Homology, Amino Acid
  • Sequence Analysis, DNA
  • Recombinases
  • Protein Structure, Secondary
  • Protein Engineering
  • Protein Conformation
  • Mutagenesis
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Gaj, T., Mercer, A. C., Gersbach, C. A., Gordley, R. M., & Barbas, C. F. (2011). Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proceedings of the National Academy of Sciences of the United States of America, 108(2), 498–503. https://doi.org/10.1073/pnas.1014214108
Gaj, Thomas, Andrew C. Mercer, Charles A. Gersbach, Russell M. Gordley, and Carlos F. Barbas. “Structure-guided reprogramming of serine recombinase DNA sequence specificity.Proceedings of the National Academy of Sciences of the United States of America 108, no. 2 (January 2011): 498–503. https://doi.org/10.1073/pnas.1014214108.
Gaj T, Mercer AC, Gersbach CA, Gordley RM, Barbas CF. Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proceedings of the National Academy of Sciences of the United States of America. 2011 Jan;108(2):498–503.
Gaj, Thomas, et al. “Structure-guided reprogramming of serine recombinase DNA sequence specificity.Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 2, Jan. 2011, pp. 498–503. Epmc, doi:10.1073/pnas.1014214108.
Gaj T, Mercer AC, Gersbach CA, Gordley RM, Barbas CF. Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proceedings of the National Academy of Sciences of the United States of America. 2011 Jan;108(2):498–503.
Journal cover image

Published In

Proceedings of the National Academy of Sciences of the United States of America

DOI

EISSN

1091-6490

ISSN

0027-8424

Publication Date

January 2011

Volume

108

Issue

2

Start / End Page

498 / 503

Related Subject Headings

  • Transposon Resolvases
  • Transgenes
  • Serine
  • Sequence Homology, Amino Acid
  • Sequence Analysis, DNA
  • Recombinases
  • Protein Structure, Secondary
  • Protein Engineering
  • Protein Conformation
  • Mutagenesis