Analysis of DNA mismatch repair system from Neisseria meningitidis

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Title: Analysis of DNA mismatch repair system from Neisseria meningitidis
Authors: Xue, Liang
Item Type: Thesis or dissertation
Abstract: 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.
Content Version: Open Access
Issue Date: Nov-2016
Date Awarded: Oct-2017
Supervisor: Baldwin, Geoffrey
Department: Life Sciences
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Master of Philosophy (MPhil)
Appears in Collections:Life Sciences PhD theses

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