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E.coli systems and recombination: Determinants of diversity: Overall aims ML

E.coli systems and recombination: Determinants of diversity: Overall aims ML. Nine/ten lectures with Key topics. Homologous recombination and DNA repair Role of methylation and repair . Role of Plasmids; control of replication, transfer and stability.

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E.coli systems and recombination: Determinants of diversity: Overall aims ML

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  1. E.coli systems and recombination: Determinants of diversity: Overall aims ML • Nine/ten lectures with Key topics. • Homologous recombinationand DNA repair • Role of methylation and repair. • Role of Plasmids; control of replication, transfer and stability. • Illegitimate recombination: transposons and integrons • Regulation of DNA transposition. • You should: • Have a basic grounding for further reading and other systems covered in the course (e.g pathogens). • Be able to critically read key papers in the area. • Critically assess the development of ideas to date.

  2. DNA Methylation - A common mechanism for sequence regognition • Restriction and modification systems • Discovered as Phage restriction • Type 1: recognise sequence but cut at random • hsd R, S, M in E .coli K12 • E. coli also has two other systems • mar : Recognises methylated Adenine • BUT NOT when methylated by Dam methylase • mcr : recognises methylated cytosine • BUT NOT when methylated by Dcm methylase

  3. E. coli methylases. • Cytosine methylation • dcm gene encodes cytosine methylase • protects EcoRII restriction site (by chance) • 5’-CCA/TGG-3’ (C -methylated) 5-methylcytosine • (note EcoRII restriction is UNRELATED) • Associated with a very short patch repair system not fully defined. • Over 500 sites on the E. coli chromosome

  4. E. coli methylases. • Adenine methylation • dam gene encodes adenine methylase • hemimethylated for some minutes following DNA replication • 5’-GATC-3’ (A-methylated) 6-methyl adenine • Definitely associated with MISMATCH REPAIR • dam mutants are recognised as MUTATORS • Higher frequency of spontaneous mutations as base-pair transitions

  5. Other mutator genes. • Many isolated and mapped • Many involved in mismatch repair • mutH ; mutL ; mutS and mutU involved along with dam • GATC sequence must be important here but only 256 of these sites on the E. coli chromosome • Two models proposed for mismatch repair

  6. MutS MutL ssb MutU G GATC GATC GATC GATC GATC GATC GATC GATC GATC GATC CTAG CTAG CTAG CTAG CTAG CTAG T CTAG CTAG MutH G C Synthesis GATC Degraded Mismatch repair model. Mismatch repaired

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