Recognition and processing of branched DNA substrates by Slx1–Slx4 nuclease

Abstract

Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3′ end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures.

Coordinated roles of SLX4 and MutSβ in DNA repair and the maintenance of genome stability

“…SLX1 interacts with the C-terminal coiled-coil domain (CCD) of SLX4 (also known as the SLX1 binding domain, or SBD), and SLX1-SLX4 CCD exhibits endonuclease activity in vitro ( Gaur et al 2015 , 2019 ). In the absence of SLX4, SLX1 exhibits a weak endonuclease activity that is stimulated 500-fold by SLX4 ( Fricke and Brill 2003 ).…”

“…Structural analyses revealed the molecular basis for this SLX4-dependent activation, such that SLX1 forms a compact stable homodimer in vitro that blocks the active site of SLX1. However, in complex with the SLX4 CCD , the active site of SLX1 becomes accessible ( Gaur et al 2015 , 2019 ).…”

Structural Aspects of DNA Repair and Recombination in Crop Improvement

“…Instead, SLX1 has DNA binding patches on its surface. SLX1 uses the spatial organization of these DNA binding patches to bend the DNA substrate and identify the branching point as a flexible discontinuity in branched DNA substrates ( Figure 4D; Gaur et al, 2019). Based on the available sequences, a GIY-YIG containing, SLX1 like protein called HIGLE has been identified in Arabidopsis (Cho et al, 2017).…”

“…SLX1-SLX4 complex participates in HR and maintenance of telomeres. Biochemically, SLX1-SLX4 complex can cleave HJs, 5′ flap, replication forks, 3′ flaps, and splayed arm DNA substrates ( Fricke and Brill, 2003 ; Gaur et al, 2015 , 2019 ). Crystal structures of SLX1 and SLX1 in complex with CCD domain of SLX4 (unliganded and in complex with DNA substrate) provided insights into the regulation of SLX1 activity and substrate specificity.…”

“…Crystal structures of SLX1 and SLX1 in complex with CCD domain of SLX4 (unliganded and in complex with DNA substrate) provided insights into the regulation of SLX1 activity and substrate specificity. SLX1 comprises an N-terminal GIY-YIG nuclease domain, and a C-terminal RING domain connected by a long α-helix ( Gaur et al, 2015 , 2019 ). In fungi, SLX1 exists in a self-inhibitory homodimeric form.…”