Bound nucleotide controls the endonuclease activity of mismatch repair enzyme MutL.
DNA mismatch repair corrects mismatched base pairs mainly caused by replication error. Recent studies revealed that human MutL endonuclease, hPMS2, plays an essential role in the repair. However, there has been little biochemical analysis of the MutL endonuclease. In particular, it is unknown for what the MutL utilizes ATP binding and hydrolyzing activity. Here we report the detailed functional analysis of Thermus thermophilus MutL (ttMutL). ttMutL exhibited an endonuclease activity that decreased on alteration of Asp-364 in ttMutL to Asn. The biochemical characteristics of ttMutL were significantly affected on ATP binding, which suppressed nonspecific DNA digestion and promoted the mismatch- and MutS-dependent DNA binding. The inactivation of the cysteinyl residues in the C-terminal domain resulted in the perturbation in ATP-dependent regulation of the endonuclease activity, although the ATP-binding motif is located in the N-terminal domain. Complementation experiments revealed that the endonuclease activity of ttMutL and its regulation by ATP binding are necessary for DNA repair in vivo.
Fukui
Kenji
K
RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
Nishida
Masami
M
Nakagawa
Noriko
N
Masui
Ryoji
R
Kuramitsu
Seiki
S
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053843:DNA Mismatch Repair; D019281:Dimerization; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology*
D000255:Adenosine Triphosphate; D004247:DNA; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D003545:Cysteine