CHARACTERIZING THE C-TERMINALREGION OF PARA TO DETERMINE ITSROLE IN REPRESSOR ACTIVITYBy: Nova SyedSupervisor: Dr. Barbara...
INTRODUCTION
INTRODUCTIONBouet and Funnell, 1999
PAR+/REP- MUTANTS
SEPARATING THE H323Y+E332V DOUBLE MUTANT
FLOW CHART OF OVERALL CLONING STEPS
THE REPRESSION ASSAY                                  β-galactosidase                                                     ...
C-TERMIINAL MUTANTS DO NOT HAVE REPRESSOR ACTIVITY                                           2000                         ...
THE PARTITION ASSAY            Grow culture             overnight in            LB+Ampicillin        Plate on       M9 Glu...
H323Y AND E332V HAVE DEFECTIVE            PARTITION ACTIVITY                     Partition Activity   Repressor           ...
OVERVIEW OF PROTEIN PURIFICATION                  Harvest Cells              Lyse By Sonication                DE-52 Colum...
SDS-PAGE OF PROTEIN PURIFICATION STEPS t=0 t=120min        FrI   FrII-1   FT   WI   100-3 200-1   200-2 200-3   200-4     ...
MAP OF TRYPTIC FRAGMENTS OF PARA
E332A BINDS ATP AND ADPE332A            Wildtype ParA
ELECTROPHORETIC MOBILITY SHIFT ASSAY
E332A HAS A DAMAGED SITE-SPECIFIC DNA              BINDING ACTIVITY                  Wildtype ParA   E332A   2log DNA     ...
MAIN CONCLUSIONSH323Y, E332V and E332A all have defective repressor activity   E332V and H323Y may be compensating mutatio...
FUTURE DIRECTIONS   Further investigate the role of this C-terminal region in binding parOP   DNAseI footprinting would ...
REFERENCESBouet,Jean-Yves and Funnell, Barbara E. P1 ParA interacts with the P1 partition complex at parS and an ATP–ADP  ...
ACKNOWLEDGEMENTS Dr. Barbara Funnell       Lori Ing    James Havey
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4th Year Thesis

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  • -Accurate distribution of daughter chromosomes at cell division is essential to ensure that each cell receives a complete copy of the genome. In bacteria, the process of chromosome partition involves the separation and positioning of daughter chromosomes in each cell cycle. -Low copy number plasmids are also stably inherited via active partition systems, and these systems are responsible for directing plasmids to distinct intracellular sites. The prophage of bacteriophage P1 exists as a low copy number plasmid that is faithfully segregated by a type1a partition system. -The P1 Par system consists of two proteins, ParA and ParB, which act on a centromere-like site called parS. ParB, along with E.coli integration host factor (IHF), binds to parS to form a partition complex. ParA, an ATPase is required for the proper positioning of these partition complexes. Homologs of ParA and ParB are encoded by a variety of bacterial plasmids as well as by the chromosomes of a large number of bacterial species, although not by E.coli. Several homologs have been shown to contribute to partition of the bacterial chromosomes.
  • -relevance of par system (broad context)P1 exists as a low copy number plasmid that is faithfully segregated by a type1a partition system. The P1 Par system consists of two proteins, ParA and ParB, which act on a centromere-like site called parS. ParB, along with E.coli integration host factor (IHF), binds to parS to form a partition complex. ParA, an ATPase is required for the proper positioning of these partition complexes. Homologs of ParA and ParB are encoded by a variety of bacterial plasmids as well as by the chromosomes of a large number of bacterial species, although not by E.coli. Several homologs have been shown to contribute to partition of the bacterial chromosomes. ParA plays at least two roles in P1 plasmid partition, a positioning one during the segregation reaction and a transcriptional one as the repressor of the par operon. Adenine nucleotide binding is necessary for both these roles. ParA binds specifically to an inverted repeat sequence in the par promoter, parOP, and this activity is stimulated strongly by adenine nucleotides. ParB also influences ParA’s activities in vitro and in vivo. It acts as a corepressor, stimulating the repressor activity of ParA, but ParB has no repressor activity on its own. In vitro, ParB stimulates ParA’s ATPase activity and its DNA-binding activity towards parOP. In the latter case, however, ParB could stimulate ParA’s DNA-binding activity only in the presence of ATP and only to a level seen with ParA (alone) in the presence of ADP. One possible explanation is that ParB stimulates ParA’s DNA-binding activity by shielding it from the negative effects of ATP hydrolysis.-The P1 plasmid is stably maintained and faithfully segregated in Escherichia coli by a type Ia partition system, which involves the action of two partition proteins, ParA and ParB, and a cis-acting centromere-like element called parS. P1 ParA is a Walker-type ATPase essential for plasmid localization, while ParB is a DNA binding protein that specifically recognizes parS. ParA is also a transcriptional repressor of the par operon, by binding to parOP. ParB serves as a co-repressor by stimulating ParA’s ATPase activity.
  • In a previous project, repressor-specific ParA mutants with an intact partition activity were isolated by PCR mutagenesis. A significant number of the mutations created were highly concentrated in a C-terminal region of ParA spanning only 17 amino acids. Although domain-swapping experiments have designated the C-terminus of ParA to interact with ParB, this specific region has been minimally characterized. Based on X-ray crystallography studies on P1 and P7 ParA, this region resides near the surface of the protein, indicating dimerization of the repressor form of ParA (which is required for binding to parOP) may be affected. This project involves characterizing a set of repressor-specific ParA C-terminal mutants that exhibit intact partition activity. Although domain-swapping experiments have designated the C-terminus of ParA to interact with ParB, this specific region (amino acids 323-340) has been minimally characterized . -figure with domainsshow where mutations (EB’s); remind audience why c-term is interesting-objective= 1 bullet
  • C-terminal mutations that will be studied in an effort to identify the function of this region of ParA, are located at amino acid position 323 and 332. Since these mutations were initially isolated as a double mutant H323Y +E332V, site directed mutagenesis has been conducted to recreate each one individually, in addition to the creation of a new E332A mutation (which completely removes the charged glutamate side chain). Thus, the intended approach of this project is to initially use molecular biology techniques to clone these individual mutants into different plasmid backgrounds for testing phenotypes in vivo using reporter-based assays, followed by protein purification and biochemical tests to determine if the mutant proteins are amenable for further experimentation.
  • C-terminal mutations that will be studied in an effort to identify the function of this region of ParA, are located at amino acid position 323 and 332. Since these mutations were initially isolated as a double mutant H323Y +E332V, site directed mutagenesis has been conducted to recreate each one individually, in addition to the creation of a new E332A mutation (which completely removes the charged glutamate side chain). Thus, the intended approach of this project is to initially use molecular biology techniques to clone these individual mutants into different plasmid backgrounds for testing phenotypes in vivo using reporter-based assays, followed by protein purification and biochemical tests to determine if the mutant proteins are amenable for further experimentation.
  • β-galactosidase assays, performed as per Miller 1992, using a parOP-parA::lacZ reporter demonstrate in vivo repressor activity of the mutants under study. Wildtype ParA +ParB serve as the positive control, indicated by complete repression. B-galactosidase level with ParA alone is similar to the absence of both ParA and ParB (which serve as negative and technical control, respectively). Error bars represent variation in triplicate readings in this experiment (one standard deviation).
  • The tester strain DH5lacI:tclacZ+(pBEF240) was used to test for partition activity by blue/white colour screening on M9-Xgal media. A single colonies of DH5 lacI::tclacZ+ cells containing pBEF240, and pEF5 derivative of mutant and wildtype parA were picked from an LB agar plate containing ampicillin and chloramphenicol, diluted into LB with ampicillin and grown overnight at 37°C. This is because the parS containing pBEF240 plasmid also encodes a gene for chloramphenicol resistance. Samples taken from the beginning and end of the growth period were plated for single colonies on M9 plates containing X-gal and Glucose. The resulting colonies that had retained pBEF240 would be expressing lacI and therefore appear white on M9-Xgal, because β-galactosidase would not be expressed. Those unable to retain the reporter plasmid due to a defective par system would appear blue.
  • The tester strain DH5lacI:tclacZ+(pBEF240) was used to test for partition activity by blue/white colour screening on M9-Xgal media. (+) indicates some maintenance of pBEF240, by light blue colonies. (++) indicates >99% maintenance, by white colonies.the single mutants H323Y and E332V were observed to have compromised partition activity. Surprisingly, the E332V mutation exhibited an intermediate par- phenotype in comparison to H323Y and E332A.
  • As shown in Figure 8, E332A mutant protein was successfully purified by DE-52 and Ni+2–sepharose column chromatography (steps shown in Methods section). T=0min and t=120min mark lanes with total cells, ~0.1 A600 units of BL21(λDE3) (pLysS) cells before (t=0min) and two hours after (t=120) induction. The protein fraction lanes are FrI (crude lysate, 14ug), FrII-1(DE-52 eluate, 16.4ug), FT (flow through of Ni+2 column, 15ug), WI (first wash of Ni+2 –Sepharose column with 20mM imidazole, 15 ug), and eluates using increasing concentrations of imidazole (100-3 is 100mM, 200-1 is 200mM imidazole, etc). The amounts of protein loaded in fractions 100-3 to 200-4 are 3.5ug, 3.ug, 14ug, 4.8ug, and 0.8ug, respectively. ParA was identified as the major band at approximately 46 kDa (its predicted size from DNA sequence without a (His)6 tag is 44 kDa.
  • As shown in Figure 8, E332A mutant protein was successfully purified by DE-52 and Ni+2–sepharose column chromatography (steps shown in Methods section). T=0min and t=120min mark lanes with total cells, ~0.1 A600 units of BL21(λDE3) (pLysS) cells before (t=0min) and two hours after (t=120) induction. The protein fraction lanes are FrI (crude lysate, 14ug), FrII-1(DE-52 eluate, 16.4ug), FT (flow through of Ni+2 column, 15ug), WI (first wash of Ni+2 –Sepharose column with 20mM imidazole, 15 ug), and eluates using increasing concentrations of imidazole (100-3 is 100mM, 200-1 is 200mM imidazole, etc). The amounts of protein loaded in fractions 100-3 to 200-4 are 3.5ug, 3.ug, 14ug, 4.8ug, and 0.8ug, respectively. ParA was identified as the major band at approximately 46 kDa (its predicted size from DNA sequence without a (His)6 tag is 44 kDa.
  • Mapping tryptic fragments of ParA: The cleavage sites, derived from N-terminal sequence analysis, are shown. The N-termini of fragments A, B, and E start at Met17, of fragments C and D at His165, and of FL (full-length) at Met1. The positions of the right-most cleavage site and of the C-termini of each fragment are estimated from the fragment sizes.
  • Figure 9: E332A can bind nucleotide, which results in protection from digestion. Trypsin proteolysis of wild type ParA and E332A without nucleotide (lanes 2,3,9, and 10), with 2mM ATP (lanes 4,5, 11 and 12), and with 2mM ADP (lanes 6,7, 13 and 14). Each reaction consisted of 2.5ug of ParA and varying amounts of trypsin (+ indicates 1:200, ++ indicates 1:100 trypsin to ParA ratio). All reactions were incubated at room temperature for 2 hours. Protein markers indicate size in kDa.
  • Electrophoretic mobility shift assay of a parOP containing 341bp DNA fragment, by increasing amounts of wildtype and mutant protein (58ng, 120ng, 230ng, and 460ng) demonstrates that E332A has compromised ability to specifically bind parOP . Binding assays were conducted in TBM buffer, containing 2mM ADP. Figure 10: E332A has compromised ability to specifically bind parOP. Electrophoretic mobility shift assay of a 341bp DNA fragment containing parOP, with increasing amounts of wildtype and mutant protein (58ng, 120ng, 230ng, and 460ng). Binding assays were conducted in TBM buffer, containing 2mM ADP and incubated at 30°C for 15min.
  • E332V and H323Y mutations are compensating for each other in the partition form of ParA, since the double mutant has partition activity but the single mutants do not.Gel-shift assays demonstrate that the E332 position is required in the repressor form of ParA, as removal of this residue significantly reduces its ability to bind parOP . Since this mutant stably bound both ATP and ADP, the E332 residue may be playing an essential role in specific DNA binding.Future experiments would include DNA footprinting and examination of whether dimerization of the repressor form of ParA is being affected.In order to further elucidate the cause of the repressor- phenotype of E332A, gel-shift assays were conducted in the presence of ADP. As shown in Figure 10, the 341bp parOP DNA fragment did not shift with E332A, compared to a significant band shift with only 120 ng of wild type ParA. The binding reaction for these assays consisted of a 15 minute incubation time, and the gels were stained with SYBR Green Fluorescent Nucleic Acid stain for 30 min. If the cooperativity of ParA binding to parOP is affected, a longer incubation and staining time may demonstrate a greater shift. As the tryptic digests demonstrated functional ADP binding by E332A, the repressor – phenotype is most likely to be attributed to its ability to specifically bind parOP. Non-specific DNA binding is unlikely to be affected, as this would be required for partition, and the stability assays suggested that E332A is partition+.
  • This putative C-terminal DNA binding region of ParA may be involved in direct contact with parOP DNA, cooperativity -how specific binding to ParOP is affected by the E332A mutation, supporting the results of the gel-shift experiments. DNAseI footprinting is an especially good indicator of cooperativity in ParA binding to parOP. Thus, if minimal protection of parOP is observed even at high concentrations of ParA, E332A may be defective in repression due to a lack of cooperativity and the mutation may be hindering ParA-ParA interactions. Although the E332A mutation does not lie on the dimerization interface of ParA, further investigation is required to determine if the mutation is causing the repressor form of ParA dimer to dissociate. Experiments to examine this possibility include analysis through a non-denaturing protein gel, and fluorescence resonance energy transfer. this future work may be initiated by determining if the single E332V and H323Y mutants can bind and hydrolyze ATP. If so, experimentation can move forward to their binding affinity to non-specific DNA (e.g. filter binding assays).
  • 4th Year Thesis

    1. 1. CHARACTERIZING THE C-TERMINALREGION OF PARA TO DETERMINE ITSROLE IN REPRESSOR ACTIVITYBy: Nova SyedSupervisor: Dr. Barbara Funnell
    2. 2. INTRODUCTION
    3. 3. INTRODUCTIONBouet and Funnell, 1999
    4. 4. PAR+/REP- MUTANTS
    5. 5. SEPARATING THE H323Y+E332V DOUBLE MUTANT
    6. 6. FLOW CHART OF OVERALL CLONING STEPS
    7. 7. THE REPRESSION ASSAY β-galactosidase +ortho-Nitrophenyl-β-galactoside galactose ortho-nitrophenol
    8. 8. C-TERMIINAL MUTANTS DO NOT HAVE REPRESSOR ACTIVITY 2000 1800 1600 1400B-Galactosidase Activity (Miller Units) 1200 1000 800 600 400 200 0 WT ParA+ParB H323Y+E332V H323Y +WTParB E332V +WTParB E332A +WTParB WT ParA noParA no ParB +WTParB
    9. 9. THE PARTITION ASSAY Grow culture overnight in LB+Ampicillin Plate on M9 Glu Xgal galactose 5-bromo-4-chloro- 5,5-dibromo-4,4-dichloro-indigo 3-hydroxyindolepar+ par¯
    10. 10. H323Y AND E332V HAVE DEFECTIVE PARTITION ACTIVITY Partition Activity Repressor Actvity H323Y - - - Blue E332V + - + Light Blue ++ White E332A ++ - H323Y+E332V ++ - Wildtype ++ + pBR322 - -(no ParA, no ParB)
    11. 11. OVERVIEW OF PROTEIN PURIFICATION Harvest Cells Lyse By Sonication DE-52 Column Ni+2-Sepharose Column Wash with low [Imidazole] Wash Buffer Elute with high [Imidazole] Elution Buffer Check [Protein] by Bradford Assays
    12. 12. SDS-PAGE OF PROTEIN PURIFICATION STEPS t=0 t=120min FrI FrII-1 FT WI 100-3 200-1 200-2 200-3 200-4 175kDa 80 58 46 30 25 17 7
    13. 13. MAP OF TRYPTIC FRAGMENTS OF PARA
    14. 14. E332A BINDS ATP AND ADPE332A Wildtype ParA
    15. 15. ELECTROPHORETIC MOBILITY SHIFT ASSAY
    16. 16. E332A HAS A DAMAGED SITE-SPECIFIC DNA BINDING ACTIVITY Wildtype ParA E332A 2log DNA No Ladder ProteinparOP 300bp 200 100
    17. 17. MAIN CONCLUSIONSH323Y, E332V and E332A all have defective repressor activity E332V and H323Y may be compensating mutations in the partition form of ParA E332A mutant binds nucleotide E332A is defective in specific binding of parOP
    18. 18. FUTURE DIRECTIONS Further investigate the role of this C-terminal region in binding parOP DNAseI footprinting would not only confirm gel-shift results, but also elucidate whether cooperative binding by ParA is affected. Native gels and FRET to determine whether dimerization of ParA is compromised The interaction between E332V and H323Y which allows only the double mutant to maintain its partition activity needs to be further examined. This would require designing a more sophisticated assay to test partition activity.
    19. 19. REFERENCESBouet,Jean-Yves and Funnell, Barbara E. P1 ParA interacts with the P1 partition complex at parS and an ATP–ADP switch controls ParA activities. 1999. The EMBO Journal Vol.18 No.5 pp.1415–1424Davey MJ, Funnell BE. Modulation of the P1 plasmid partition protein ParA by ATP, ADP, and P1 ParB. J Biol Chem. 1997 Jun 13;272(24):15286-92.Dunham TD, Xu W, Funnell BE, Schumacher MA. Structural basis for ADP-mediated transcriptional regulation by P1 and P7 ParA. EMBO J. 2009 Jun 17;28(12):1792-802. Epub 2009 May 21.Fung E, Bouet JY, Funnell BE. Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis. EMBO J. 2001 Sep 3;20(17):4901-11.Funnell, B, & Slavcev, RA. (2004). Plasmid biology: Ch 5: Partition systems of bacterial plasmids. Washington, DC: ASM Press.Surtees, J. A., & Funnell, B. E. (2003). Plasmid and Chromosome Traffic Control: How ParA and ParB Drive Partition. Current Topics in Developmental Biology Vol. 56(pp. 145-174.). Elselvier Inc.
    20. 20. ACKNOWLEDGEMENTS Dr. Barbara Funnell Lori Ing James Havey

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