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PMID:22223640

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Citation

Lin, JL, Nakagawa, A, Lin, CL, Hsiao, YY, Yang, WZ, Wang, YT, Doudeva, LG, Skeen-Gaar, RR, Xue, D and Yuan, HS (2012) Structural insights into apoptotic DNA degradation by CED-3 protease suppressor-6 (CPS-6) from Caenorhabditis elegans. J. Biol. Chem. 287:7110-20

Abstract

Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg(2+)-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αβ topology with the two ββα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg(2+)-bound active site. Mutations of four aromatic and basic residues: Phe(122), Arg(146), Arg(156), and Phe(166), in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.

Links

PubMed PMC3293555 Online version:10.1074/jbc.M111.316075

Keywords

Amino Acid Motifs; Amino Acid Substitution; Animals; Apoptosis/physiology; Caenorhabditis elegans/enzymology; Caenorhabditis elegans/genetics; Caenorhabditis elegans Proteins/chemistry; Caenorhabditis elegans Proteins/genetics; Caenorhabditis elegans Proteins/metabolism; Crystallography, X-Ray; DNA, Helminth/chemistry; DNA, Helminth/genetics; DNA, Helminth/metabolism; Endodeoxyribonucleases/chemistry; Endodeoxyribonucleases/genetics; Endodeoxyribonucleases/metabolism; Hydrolysis; Mitochondrial Proteins/chemistry; Mitochondrial Proteins/genetics; Mitochondrial Proteins/metabolism; Models, Molecular; Mutation, Missense; Structure-Activity Relationship

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CAEEL:NUCG

enables

GO:0004520: endodeoxyribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:NUCG

enables

GO:0004521: endoribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:NUCG

involved_in

GO:0006401: RNA catabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

CAEEL:NUCG

involved_in

GO:0000737: DNA catabolic process, endonucleolytic

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

CAEEL:NUCG

enables

GO:0043565: sequence-specific DNA binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:NUCG

involved_in

GO:0006309: apoptotic DNA fragmentation

ECO:0000315: mutant phenotype evidence used in manual assertion

WB:WBVar00242728

P

Seeded From UniProt

complete

CAEEL:NUCG

enables

GO:0042803: protein homodimerization activity

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q95NM6

F

Seeded From UniProt

complete


See also

References

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