Massively parallel single-amino-acid mutagenesis.


Abstract

Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.

Submission Details

ID: 7wvUDLZY3

Submitter: Shu-Ching Ou

Submission Date: July 18, 2018, 5:55 p.m.

Version: 1

Publication Details
Kitzman JO;Starita LM;Lo RS;Fields S;Shendure J,Nat Methods (2015) Massively parallel single-amino-acid mutagenesis. PMID:25559584
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AW6 1998-04-15 GAL4 (CD), NMR, 24 STRUCTURES
1HBW 2001-05-10 Solution nmr structure of the dimerization domain of the yeast transcriptional activator Gal4 (residues 50-106)
3COQ 2008-07-01 2.4 Structural Basis for Dimerization in DNA Recognition by Gal4
1D66 1992-03-06 2.7 DNA RECOGNITION BY GAL4: STRUCTURE OF A PROTEIN/DNA COMPLEX
3BTS 2008-03-04 2.7 Crystal structure of a ternary complex of the transcriptional repressor Gal80p (Gal80S0 [G301R]) and the acidic activation domain of Gal4p (aa 854-874) from Saccharomyces cerevisiae with NAD

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Regulatory protein GAL4 P04386 GAL4_YEAST