Junction resolving enzymes use multivalency to keep the Holliday junction dynamic

Abstract

Holliday junction (HJ) resolution by its resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch-point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single molecule fluorescence resonance energy transfer analysis of resolving-enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially-dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving-enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.

A bacterial resolvase first exploits, then constrains intrinsic dynamics of the Holliday junction to direct recombination

“…2). Occasional branch migration of the HJ was detected as a variation in FRET dynamics by visual inspection, as observed before 11 .…”

“…2a and 2b); in particular, a new mid-FRET state appears (E FRET = 0.4±0.1) that lies between the high-FRET iso-I (E FRET = 0.64±0.13) and the low-FRET iso-II (E FRET = 0.17±0.1). This state is similar to the planar open conformation of the HJ in the absence of divalent metal ions 10,11 , and is henceforth termed "open", or Op. Upon replacing Mg 2+ with Ca 2+ , this Op state disappears, leaving only iso-I (E FRET = 0.68±0.09) and iso-II (E FRET = 0.15±0.08, Fig.…”

“…Numerous in vitro studies with synthetic DNA have shown that RuvC recognizes a HJ structurally, but in sequence-independent manner. More specifically, in a recent study Zhou et al 11 showed that a RuvC-bound HJ retains its intrinsic fluctuations [12][13][14][15] between two alternatively stacked, X-shaped conformational isomers (iso-I and iso-II), while an intermittent, multivalently bound, yet partially dissociated (PD) RuvC allows the HJ to undergo nearly unimpeded conformer exchange as well as branch migration 11 . However, it has remained unclear how RuvC achieves its remarkable cleavage specificity for the cognate 5'-ATT ↓ X-3' sequence (X = G/C; ↓ = cleavage site) to ensure proper control over chromosome segregation 1,3,[16][17][18] .…”

“…More than twelve amino acids from each monomer of RuvC form redundant contacts with the junction 6 , allowing the resolvase to remain loosely associated in the PD state so that the HJ can transition between isomers iso-I and iso-II as well as branch migrate (Fig. 3) 11 . Once the cognate cleavage sequence is approaching the enzyme's catalytic core near the junction via this rapid sampling, charged amino acids such as Arg76 in the core of RuvC stabilize the G-C base of the consensus cleavage sequence via amino acidnucleo base cation-pi interactions 6 .…”

Freedom of movement

“…Free HJs are highly dynamic, but crystal-structure analyses of resolvase-HJ complexes show extensive protein-DNA interactions that appear to block any movement, unless the complex dissociates 2 . Zhou et al 3 now use single-molecule experiments to reveal nearly unencumbered HJ dynamics in the enzyme-bound form. This unexpected behavior was observed for HJs bound by a range of viral, bacterial, fungal and human enzymes.…”