Neisseria meningitidis (N. meningitidis) mismatch repair (MMR) proteins MutS
(NmMutS) and MutL (NmMutL) are reported to be involved in phase variation,
which helps this bacterial pathogen survive in the human host. The MMR
system is responsible for the correction of mismatch errors that occur during
DNA replication. Unlike the extensively studied E. coli system, MMR from N.
meningitidis lacks MutH endonuclease and is poorly understood in terms of its
coordination and mechanism of strand discrimination. This project aims to
investigate the function of MMR enzymes of N. meningitis via in vitro assays.
As an irreplaceable polymerase in MMR, DNA Polymerase III holoenzyme
(DNA Pol III HE) was reconstituted from individual components in good yield in
vitro. Furthermore, in gel shift assays, NmMutS exhibited weak mismatch
binding, while E. coli MutS bound a mismatched DNA with high affinity. NmMutL
non-specifically bound to DNA duplexes without mismatch discrimination. The
NmMutS and NmMutL combination also bound to non-specific DNA that pointed
towards a downstream activity of NmMutL, rather than mismatch recognition.
NmMutL carried out Mn2+-dependent nuclease activity with low mismatch
specificity in in vitro assays, and exhibited different degrees of digestion on two
strands of the mismatched DNA in the presence of Mg2+. The nuclease assay
also suggest that NmMutS may enhance the mismatch specific nuclease
activity of NmMutL. The addition of ATP inhibited the nuclease activity of
NmMutL under different mental ion conditions. This study assessed roles and
interactions of NmMutS and NmMutL in the initiation of N. meningitidis MMR
system which coordinated with ATP and metal ions in vitro.