Nicole A. Becker and L. James Maher

BIOCHEMISTRY

Significance

Double-stranded DNA is one of the stiffest polymers in biology, resisting both bending and twisting over hundreds of base pairs. However, tightly bent DNA loops are formed by proteins that turn off (repress) genes in bacteria. It has been shown that “architectural” proteins capable of kinking any DNA molecule without sequence preference facilitate this kind of gene repression. The mechanism of this effect is unknown for DNA loops involving the well-known Escherichia coli lac repressor. Here we adapt high-resolution protein-mapping techniques to show that an architectural protein directly binds tightly looped DNA to facilitate gene repression by the lac repressor.

Abstract

Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

See: http://www.pnas.org/content/112/23/7177.abstract.html?etoc

PNAS June 9, 2015 vol. 112 no. 23: 7177-7182

Fig. 4.

Summary of high-resolution protein binding data and model. (A) Four tested lac promoter constructs with the indicated lac operators and promoter elements showing protein binding sites identified by ChIP-exo-LMPCR (LacI, circles; σ⁷⁰, triangles; Nhp6A, squares near TG/CA dinucleotide, underlined), and by ChEC-LMPCR (Nhp6A-MNase, crosses). (B) Model of LacI loop showing operators (cyan and blue), promoter elements (magenta), and Nhp6A cleavage sites (red) identified by ChEC-LMPCR near the TG/CA dinucleotide (black) proposed as the kinked binding site. (Inset) Illustration of plausible intercalation of Nhp6A methionine 29 at the TG/CA dinucleotide based on [PDB ID code 1J5N (25)] after rotation of the complex by 180° about a vertical axis.