The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL.
The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn(2+), Mg(2+) or Ca(2+) ions, unlike human MutLalpha which shows endonuclease activity only in the presence of Mn(2+). We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460-658 exhibits Mn(2+)-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX2EX4E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX2EX4E motif.
Duppatla
Viswanadham
V
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Bodda
Chiranjeevi
C
Urbanke
Claus
C
Friedhoff
Peter
P
Rao
Desirazu N
DN
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004247:DNA / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D000070956:MutL Proteins; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D004247:DNA; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D045643:DNA Repair Enzymes