1. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. E.coli systems and recombination: Determinants of diversity: Overall aims ML Ten lectures with Key topics. Homologous recombination and 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. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. References http://www.blackwellpublishing.com/trun/ Molecular Genetics of Bacteria Dale and Park Dale. Paperback 352 pages (15 January, 2004) Publisher: John Wiley and Sons Ltd; ISBN: 047085085X Fundamental Bacterial Genetics Janine Trempy, Nancy Trun Janine TrempyNancy Trun Paperback 300 pages (2003) Publisher: Blackwell Science (UK); ISBN: 0632044489 http://www.blackwellpublishing.com/trun/artwork/Animations/Recombination/recombination.html

3. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Role of Plasmids; control of replication, transfer and stability. Review of DNA replication Errors in replication and their reversal Plasmid replication initiation and its control ColE1 and R1 as examples Plasmid segregation, recombination and stability Control of plasmid transfer Mobilisation functions Plasmid structure and evolution Antibiotic resistance and catabolic functions You should be able to discuss the importance of negative control of plasmid functions and their critical importance in the evolution of diverse microbial genomes

4. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Overview of DNA replication 5’ 3’ 5’ 3’ Helicase Uracil glycosylase PolIII Prepriming proteins Ssb Primer DNA Pol I Ligase

5. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Proofreading DNA polymerase AG or CT Mismatch Polymerase Removal of mismatch 3’ - 5’ digestion by polymerase Polymerisation in 5’ - 3’ direction

6. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Initiation of Chromosome replication 3 x 13 mers 4 x 9 mers oriC Hu ATP DnaA Dna B,C ATP Prepriming complex Replication

7. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Plasmid replication and control ColE1 as an example : extensively studied Different initiation mechanism to chromosome No plasmid encoded Rep protein needed to initiate replication Generally a plasmid only has to reveal an origin of replication within the cell and “hijack” the cell’s replication mechanism. But it must not over replicate too many copies and damage the cell’s functions. THREE initiation strategies are known. 1. Mimic chromosome origin as on previous slide e.g F, R1, phage P1 and 2. Plasmid encoded Rep protein initiator - ss Nick - ss template for synthesis e.g. pT181 in Staph. aureus (also in other Gm+ves) (Summers 1986) 3. Primer RNA initially synthesised from constitutive promoter. E.g ColE1

8. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. ColE1 replication oriV Small Plasmid - 4.3 Kb replication UNIDIRECTIONAL Approx’ 15 copies per cell Encodes colicin E1 and… ONE protein required in replication INITIATION OF SYNTHESIS oriV start RNA II primer formed first Starts 555 nt BEFORE oriV Synthesis catalysed by HOST DNA dependent RNA Polymerase Normally used in gene transcription HOST DNA replication mechanisms then take over MUST be a mechanism for negative regulation also.

9. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. ColE1 replication cont…….. 3’ 5’ 3’ oriV L or Light strand H or Heavy strand Poly U or Poly G tails / CsCl density grad’ centrifugation New L Strand synthesised first ONLY 580 bps needed for plasmid to replicate in host cell - 555 RNAII RNAI -445 RNA Polymerase RNAII starts at -555 on H strand

10. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. ColE1 replication cont…….. -445 - 555 3’ -445 Transcription beyond oriV Hybridisation at/near oriV - 555 3’ RNAaseH cleaves at oriV 2/3 bp accuracy -445 DNA PolI begins synthesis on primer RNA ONLY 580 bps needed and ONLY 13 after oriV

11. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. ColE1 replication cont…Chain elongation.. New H - strand synthesis Begins after initial L-strand formation Discontinuous in 5’ to 3’ direction as Okazaki fragments Fragments about 1000 bps long initiate on short RNA primers DnaB forms secondary structure in ss DNA Allows initiation by primase DnaG DNA PolI extends primer Continued L - strand synthesis DNA PolI continues for about 500 bps after initiation DNA PolII and primosome then take over L- strand extended as leading strand synthesis

12. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Control of ColE1 replication Replication control in relation to host cells growth rate? E. coli may replicate as slowly as 12h in gut and up to 40min in lab’! Efficient ColE1 maintenance. Negative control of replication initiation - low copy number TWO products involved Mutational studies on ColE1 RNA I and Rom (Rop) protein RNA I is ANTISENSE message Binds RNA II primer to prevent RNAase H processing Binding enhanced by Rom protein Cloning vectors (based on pMB1 related to ColE1) have no rom gene Copies may exceed 200-300 per cell !

13. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Control of ColE1 replication cont…... ANTISENSE RNA I binding to complementary RNA II NOT a simple base pairing mechanism. SECONDARY STRUCTURE is important Mutational studies reveal regions in the RNA I molecule necessary for control 3’ 5’ Rom GGUAGGGUAG GAGA Necessary for antisense binding RNA I secondary structure

14. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Summary of ColE1 replication control +600 +400 -500 Rop/Rom Dimer 5’ 3’ 5’ 3’ RNAase cut 3’ oriV

15. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Large conjugative plasmid replication control ColE1 is a small NON-CONJUGATIVE plasmid Many others are much larger and CONJUGATIVE or TRANSMISSIBLE (NOTE ColE1 can be transferred by mobilisation functions) Larger plasmids often present as 1 or 2 copies per cell There may be two possible ori sites Replication is often bidirectional The replication of closely related plasmids R1, R100 and R6-5 studied in detail THESE HOWEVER REPLICATE UNIDIRECTIONALLY R1 and R100 are approx’ 90Kb in size All but 2 Kb can be removed without blocking replication Must contain the oriV site Regulation differs from ColE1 regulation

16. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Regulation of R1 replication copB copA tap repA oriV 5’ 3’ 5’ Repressor RNA I antisense RNA II Shine-Delgarno repA RepA initiates replication Tap copA and copB mutations lead to x 10 increase in copy number Tap ia a transcriptional activator protein repA mutants fail to replicate RepA doubles for host Pol1

17. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Regulation of other plasmid replicons F plasmid Similar to R1 but does not depend upon RNA/RNA interactions - Phage replication Action of Cro O and P proteins CroO is an autorepressor P1 - prophage and some other plasmids ori (incC) region “HANDCUFFED” to incA region after replication Multiple 19 mer repeats RepA binding involved Works in cis and trans repA ori incA RepA repA HANDCUFFED NO repA transcript Also works in trans linking TWO P1 DNA molecules

18. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Plasmid segregation and stability INCOMPATIBILITY Plasmids with similar replication control are unstable Belong to the same incompatibility (Inc) grouping Or Incompatibility may be related to similar partitioning functions See below RANDOM PARTITIONING High copy number plasmids show random partitioning Recombination to multimers Multimers unstable Must be resolved ACTIVE PARTITIONING Proteins make sure that copies of plasmids are segregated on cell division SopA and SopB in F plasmid ParA and ParB in P1 phage

19. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Resolution of multimeric plasmids High copy number plasmids should be stable However vector plasmids such as pUC, pBR322 etc.. are very unstable particularly in rec+ hosts or sbcA mutants MULTIMERS formed easily and not resolved However ColE1 is stable Why? It has a 240bp sequence called cer that is needed for stability Similar to resolution site on Tn3 family of transposons Acted on in trans by XerB and XerC proteins Host encoded Usually act on dif sequence to resolve chromosome after replication

20. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Resolution of multimeric plasmids + 240bp cer sequence C D Xer C and Xer D Chromosomally encoded D C Form Dimer D C

21. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Active partitioning of plasmids pSC101 par region 375 bp region Binding site for DNA gyrase F1 and P1 partitioning sopA and B parA and B sopA/parA ParB/SopB ParB/SopB for a dimer Bind to sopA / sopB Associate with cell membrane and accurately segregate

22. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Regulation of plasmid transfer Will consider F and related R plasmids Then mobilisation of colE1 F Plasmid 94.5 Kb oriT Chromosomal integration IS3, IS2,  33 Kb transfer genes region oriV and rep region Very complex transfer system t.ra genes transcribed from 4 promoters in traJ region

23. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Regulation of plasmid transfer cont…. Antisense RNA involved F System tra genes traJ finP finP cannot bind IS3 finO BLOCKED finP tra genes traJ finO Binding and repression R100 System

24. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Plasmid Mobilisation Many plasmids NOT conjugative But CAN be MOBILISED by transfer functions in other plasmids ColE1 a good example mob and bom functions involved mob genes on plasmid substitute for tra genes Act on bom sequence as for oriT NO F plasmid present bom mob F plasmid present After nicking of bom site Transfer substrate is present F transfer functions transfer the col plasmid Bom site nicked by Mob proteins but NO transfer