Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates

Abstract

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.

Resolution of the Holliday junction recombination intermediate by human GEN1 at the single-molecule level

“…A previous study demonstrated that GEN1 initially binds the HJ as a monomer (34). Since we always observed the resolution within the lifetime of the GEN1-HJ complex, we propose that GEN1 monomer distorts the HJ and remains firmly bound until a dimer is formed and full resolution of the HJ takes place.…”

“…The incision requires the coordination of two active sites contrary to a mechanism where a single nick takes place, followed by protein dissociation, reassociation, then counter nicking (33). The inactivity of GEN1 monomer on HJ is supported by the observation that GEN1 monomer binds HJs more tightly than its dimer and that excess HJ decreases the cleavage rate by sequestering the monomer from solution (34). Further evidence suggests that even with the decoupling of the two incisions, either by using an active subunit of the dimer associated with a catalytically inactive partner or a non-cleavable phosphorothioate cleavage site, the first incision still seems to require GEN1 dimerization (33).…”

“…Previous biochemical characterizations highlighted the importance of dimer formation in coordinating the two incisions at the HJ (27,33,34). However, it remains unclear how GEN1 binds the HJ, coordinates the two incisions, and ensures its full resolution.…”

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

“…The attained temporal resolution can be within microseconds to several milliseconds . Understanding how GEN1 binds the HJ, coordinates the two incisions, and ensures its full resolution have all been under intensive study 21,22,23,25,26,27,28,29,30 .…”

A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1

“…There was some indication of activity on other branched species, but the enzyme was most active on four-way junctions. By contrast, GEN1 from Drosophila was found to cleave a wider range of branched substrates ( 37 ), and significantly those studies were performed in the presence of K + ions. Given the importance of K + ions in the activity and structure of the other FEN/EXO family members we sought to re-examine both the protein structure and substrate range of Ct GEN1 in the presence of other monovalent ions.…”