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. 2013 Apr;41(7):4118-28.
doi: 10.1093/nar/gkt085. Epub 2013 Feb 13.

Quantitative analysis of TALE-DNA interactions suggests polarity effects

Joshua F Meckler  1 , Mital S Bhakta, Moon-Soo Kim, Robert Ovadia, Chris H Habrian, Artem Zykovich, Abigail Yu, Sarah H Lockwood, Robert Morbitzer, Janett Elsäesser, Thomas Lahaye, David J Segal, Enoch P Baldwin
Affiliations

Quantitative analysis of TALE-DNA interactions suggests polarity effects

Joshua F Meckler et al. Nucleic Acids Res. 2013 Apr.

Abstract

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN >> NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

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Figures

Figure 1.
Figure 1.
Affinity and transcriptional activation data for several AvrBs3 variants. (A) Schematic of a TALE polypeptide showing the 18 RVD-containing repeats with N- and C-terminal flanking regions. The ‘0 repeat’ is shown in white. The numbers indicate the lengths of the N- and C-terminal extensions outside the repeat region used in the different constructs described in this work. A comprehensive survey of N- and C-terminal boundaries used in previous TALE studies is given in Supplementary Figure S1. (B) RVD amino acid composition of AvrBs3 (first row), along with the sequence of a natural DNA target, Bs3 (third row), and the consensus AvrBs3 site, UPA (2) (fourth row). The RVD composition of the dAvrBs3 variant, which contains only the standard NI, HD and NG RVDs and no mismatches to the Bs3 box target site, is also shown (second row). AvrBs3 RVDs that are ‘non-standard’ or mismatched to Bs3, and the corresponding RVDs in dAvrBs3, are underlined. The UPA site bases that differ from Bs3 are also underlined. (C) EMSA and ATF activation data were obtained as described in Materials and Methods. Target site sequences and RVD compositions are listed in Supplementary Tables S2 and S3. The affinity of Zif268 was measured in TALE 1× binding buffer. Zif268 affinity measured in a standard zinc-finger binding buffer (16) was more typical, 11 ± 4 nM.
Figure 2.
Figure 2.
Affinity and transcriptional activation data of 15 dTALEs. Comparison of (A) EMSA affinity constants for 15 dTALEs for their cognate DNA targets (KA = 1/KD, vertical axis, logarithmic scale), and (B) fold activation in an ATF assay. Guest RVD types (horizontal axis) and host contexts (I, white; II, gray; III, black bars) are indicated. Data were taken from Table 1. (C) Comparison of NN RVD interaction with G and A. EMSA affinities (left panel) and ATF fold activation (right panel) are shown for NN RVD TALE proteins with corresponding G and A DNA targets. Numerical data are given in Supplementary Table S4.
Figure 3.
Figure 3.
Natural and designed TALEs show differential dependence on 5′-T. The 5′ base of the Bs3 box target site affects the (A) affinity (KA = 1/KD, linear scale) and (B) fold activation of the natural AvrBs3111-42 (black bars) more dramatically than for a designed dAvrBs3111-42 (white bars) (see Figure 1B for RVD compositions).
Figure 4.
Figure 4.
Polarity effects of truncating substitutions at the 5′ and 3′ ends of the target site. (A) The most frequent binding motif produced by the Bind-n-Seq target site selection assay for AvrBs3254-267 is aligned below the expected UPA target site. The graph below the motif shows the enrichment of 6-mers corresponding to the 5′ end of the target site, but no enrichment of 3′ 6-mers, in the protein-bound DNA pool. (B) EMSA target sets used to test truncations (underlined) of the Bs3 box binding site are shown using IUPAC nomenclature. Target sets for the III-HDp and III-NGp are provided in Supplementary Table S2. (C, D, F and H) EMSA data are expressed as a percentage of affinity retained, compared with the 19-bp substrate, when the indicated protein binds the corresponding site with the indicated number (horizontal axis) of either 5′ (blue) or 3′ (yellow) truncated bases. (E, G and I) ATF reporter assay data are expressed as fold activation when the indicated protein binds the indicated truncated site. The activation level using the wild-type target site is shown (dashed line). Numerical data are provided in Supplementary Table S6.
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