Abstract
Holliday junctions (HJs) are key DNA intermediates in homologous recombination. They link homologous DNA strands and have to be faithfully removed for proper DNA segregation and genome integrity. Here, we present the crystal structure of human HJ resolvase GEN1 complexed with DNA at 3.0 Å resolution. The GEN1 core is similar to other Rad2/XPG nucleases. However, unlike other members of the superfamily, GEN1 contains a chromodomain as an additional DNA interaction site. Chromodomains are known for their chromatin-targeting function in chromatin remodelers and histone(de)acetylases but they have not previously been found in nucleases. The GEN1 chromodomain directly contacts DNA and its truncation severely hampers GEN1's catalytic activity. Structure-guided mutations in vitro and in vivo in yeast validated our mechanistic findings. Our study provides the missing structure in the Rad2/XPG family and insights how a well-conserved nuclease core acquires versatility in recognizing diverse substrates for DNA repair and maintenance.
Highlights
Homologous recombination (HR) is a fundamental pathway ensuring genome integrity and genetic variability (Heyer, 2015)
HR proceeds by pathways that may lead to the formation of DNA four-way junctions or Holliday junctions (HJs) that physically link two homologous DNA duplexes (Heyer, 2015; Holliday, 1964; Schwacha and Kleckner, 1995; Szostak et al, 1983)
We found that GEN1 does have the classical DNA recognition features of Rad2/XPG nucleases, and contains an additional DNA interaction site mediated by a chromodomain
Summary
Homologous recombination (HR) is a fundamental pathway ensuring genome integrity and genetic variability (Heyer, 2015). Nicked DNA strands are rejoined by endogenous ligases leading to fully restored or recombined DNA strands. This mechanism is well studied for bacterial and bacteriophage resolvases such as Escherichia coli RuvC, T7 endonuclease I, T4 endonuclease VII (Benson and West, 1994; Lilley and White, 2001). These resolvases operate as dimers and show a large degree of conformational flexibility in substrate recognition and in aligning both active sites for coordinated cleavage. T4 endonuclease VII and RuvC reach into and widen the DNA junction point whereas T7 endonuclease I binds DNA by embracing HJs at the branch point (Biertümpfel et al, 2007; Górecka et al, 2013; Hadden et al, 2007)
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