Structural Insights into Apoptotic DNA Degradation by CED-3 Protease Suppressor-6 (CPS-6) from Caenorhabditis elegans

Abstract

Background: CPS-6 (EndoG) degrades chromosomal DNA during apoptosis. Results: The crystal structure of C. elegans CPS-6 was determined, and the DNA binding and cleavage mechanisms by CPS-6 were revealed. Conclusion: The DNase activity of CPS-6 is positively correlated with its pro-cell death activity. Significance: This study improves our general understanding of DNA hydrolysis by ␤␤␣-metal finger nucleases and the process of apoptotic DNA fragmentation.

Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer

“…The purified recombinant wild-type CPS-6 (referred to as CPS-6 WT) was incubated with a 3′-end P 32 -labeled single-stranded 14-nucleotide DNA, which was degraded gradually into small fragments (<7 nucleotides) in the CPS-6 concentration-course experiments (Figure 1A). Previous studies suggest that His148 in CPS-6 functions as the general base in DNA hydrolysis, whereas Phe122 is involved in DNA binding (8). To obtain inactive CPS-6 mutants that could not digest DNA during co-crystallization experiments, we further expressed and purified three CPS-6 mutants—H148A, F122A and H148A/F122A—as described previously (8).…”

“…Previous studies suggest that His148 in CPS-6 functions as the general base in DNA hydrolysis, whereas Phe122 is involved in DNA binding (8). To obtain inactive CPS-6 mutants that could not digest DNA during co-crystallization experiments, we further expressed and purified three CPS-6 mutants—H148A, F122A and H148A/F122A—as described previously (8). Both the H148A and H148A/F122A mutants were eluted as homodimers in size exclusion chromatography, suggesting that they were assembled in a similar way as wild-type CPS-6 (Figure 1B).…”

“…We found that CPS-6 H148A/P207E was indeed eluted as a monomer in the size exclusion chromatography (Superdex 75), as compared to CPS-6 H148A that was eluted as a homodimer (Figure 5B). We further constructed another obligatory monomeric CPS-6 mutant, H148A/K131D/F132N, by altering two key interfacial residues (K131 and F132) predicted to be important for CPS-6 dimer formation based on the crystal structure of the CPS-6 H148A mutant (PDB entry: 3S5B) (8). CPS-6 H148A/K131D/F132N mutant protein also eluted as a monomer at a position identical to that of H148A/P207E (Figure 5B).…”

“…The crystal structures of EndoG have been reported, including those of Drosophila EndoG (23) and C. elegans CPS-6 (8), revealing a similar dimeric structure consisting of two active sites with a conserved DRGH sequence located within the ββα-metal motifs. Interestingly, the two ββα-metal motifs of the CPS-6 dimer are distantly located and pointed away from each other, hinting that the two protomers of the EndoG dimer likely bind to two separate DNA substrates (8).…”

“…Interestingly, the two ββα-metal motifs of the CPS-6 dimer are distantly located and pointed away from each other, hinting that the two protomers of the EndoG dimer likely bind to two separate DNA substrates (8). Recently, we further discovered that the dimeric conformation of EndoG/CPS-6 is important for the maintenance and regulation of its nuclease activity since, under oxidative conditions, EndoG/CPS-6 becomes oxidized and is dissociated into monomers with diminished nuclease activity (24).…”