Avatar
Malloutput0

0 Following 0 Followers
1
E the reporter to levels above background. If we assume that each repeat contributes a certain amount of affinity to the Bat1?Nucleic Acids Research, 2014, Vol. 42, No. 11BEBat1 interaction then fewer than 17 repeats may simply be insufficient for an interaction strong enough to lead to reporter activation. This is in accordance with results from TALE repeat arrays showing that a certain number of
1
Rminal repeats are typically inflexible, though alternative repeat -1 modules have recently been described (24,25). We tested acBat1 deletion derivatives to test if this paradigm applies to Bat1. First, we tested variants of acBat1 lacking 2 ( 18?0), 4 ( 16?0), 6 ( 14?0) or 8 ( 12?0) core repeats (Figure 5A and Supplementary Figure S10). The later half of repeat 20 and repeat +1 were retained in e
1
E the reporter to levels above background. If we assume that each repeat contributes a certain amount of affinity to the Bat1?Nucleic Acids Research, 2014, Vol. 42, No. 11BEBat1 interaction then fewer than 17 repeats may simply be insufficient for an interaction strong enough to lead to reporter activation. This is in accordance with results from TALE repeat arrays showing that a certain number of
1
Ckground (Figure 5B). This does not match expectations based on TALEs where only the cryptic N- but not the cryptic C-terminal repeats are essential for DNA binding (26). By contrast, our results suggest that the cryptic C-terminal Bat1 repeat +1, in contrast to the corresponding cryptic TALE repeat +1, makes an unexpectedly strong contribution to activity and thus should be retained for the creat
1
Nding site upstream of a uidA (GUS) reporter gene and compared to non-chimeric dTALEs (filled bars) with the same RVDs. Dashed lines separate groups of constructs all with the same RVDs and tested against the same reporter. Barred lines indicate standard deviation. Two-tailed ttests were used to compare chimeric and non-chimeric dTALEs for each reporter. A double asterisk indicates a P-value of be
1
Pplementary Figure S11. In the repeat switch whole repeats, including their native RVDs, were exchanged. This creates new interfaces between repeats but leaves RVDs in their native repeat context. If the superstructural hypothesis is correct then the repeat switch is likely to modify evolved repeat interfaces possibly yielding less active DNA-binding proteins. In the RVD switch it is only the RVDs
1
Ats are near identical and repeat order does not change the interface between repeats. Given the numerous nonRVD polymorphisms between Bat1 repeats, deletion or insertion of core repeats will always create novel repeat interfaces and should be experimentally validated before use in downstream applications. We next tested acBat1 derivatives where the 82 residues N-terminal of core repeat 1 (acBat1
1
Lymorphism in the native Bat1 as well as being relevant for the creation of Bat1 derivatives with novel specificity (dBats). We hypothesize that nonRVD polymorphisms may have two functionally relevant, non-mutually-exclusive, effects. (i) The formation of unique but functionally equivalent repeat interfaces that stabilize the superhelical structure formed by tandem-arranged repeats (4,5) (superstr