DNA mismatch repair (MMR) greatly contributes to genome integrity via the 
correction of mismatched bases that are mainly generated by replication errors. 
Postreplicative MMR excises a relatively long tract of error-containing 
single-stranded DNA. MutL is a widely conserved nicking endonuclease that 
directs the excision reaction to the error-containing strand of the duplex by 
specifically nicking the daughter strand. Because MutL apparently exhibits 
nonspecific nicking endonuclease activity in vitro, the regulatory mechanism of 
MutL has been argued. Recent studies suggest ATP-dependent conformational and 
functional changes of MutL, indicating that the regulatory mechanism involves 
the ATP binding and hydrolysis cycle. In this study, we investigated the effect 
of ATP binding on the structure of MutL. First, a cross-linking experiment 
confirmed that the N-terminal ATPase domain physically interacts with the 
C-terminal endonuclease domain. Next, hydrogen/deuterium exchange mass 
spectrometry clarified that the binding of ATP to the N-terminal domain induces 
local structural changes at the catalytic sites of MutL C-terminal domain. 
Finally, on the basis of the results of the hydrogen/deuterium exchange 
experiment, we successfully identified novel regions essential for the 
endonuclease activity of MutL. The results clearly show that ATP modulates the 
nicking endonuclease activity of MutL via structural rearrangements of the 
catalytic site. In addition, several Lynch syndrome-related mutations in human 
MutL homolog are located in the position corresponding to the newly identified 
catalytic region. Our data contribute toward understanding the relationship 
between mutations in MutL homolog and human disease.