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Seqa: Creation and Analysis of Mutants SeqA: Creation and Analysis of Mutants Master’s Thesis Presented to Biochemistry Department Brandeis University Professor Susan T. Lovett, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science Benjamin D. Hornstein May 2011 Copyright by Benjamin D. Hornstein 2011 All Rights Reserved Acknowledgements I would like to thank the members of the Lovett Lab for their support and contributions to this Thesis. Everyone there has helped create a warm and nurturing learning environment and has always been willing to lend a helping hand. In particular, I would like to thank Vincent Sutera for being my personal mentor and guiding me through this project. I would also like to thank Professor Susan Lovett for being my Thesis advisor and giving me the chance to do independent research. In addition, I would like to thank my family and friends for their encouragement and understanding. Doing research may have been a large part of my life, but I am grateful for everyone who infused sanity into the rest of my life. Finally, I would like to thank whoever continues the work on this project. Science is an ongoing process and those who come after us are just as important as those who came before. If you have any questions or concerns about the content of this thesis, feel free to contact me. iii Abstract SeqA: Creation and Analysis of Mutants A thesis presented to the Biochemistry Department Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Benjamin D. Hornstein SeqA is a protein that negatively regulates replication initiation in Escherichia coli by binding to hemimethylated GATC sites near the origin of replication. Several functions of SeqA have been well characterized, but some of the biochemical mechanisms for these functions have not been examined in detail. In order to study some of these mechanisms, we created several mutants using the Linker Scan method, as well as some site directed mutants and XL1-Red mutagenized mutants. These mutants were screened for sensitivity to DNA damaging agents. The DNA binding and dimerization abilities were then studied using flow cytometry, fluorescence microscopy, and pull down interaction experiments. This study yielded several mutants of interest, primarily using the Linker Scan method, which disturb the structure in separate locations in the protein: one elongates the N-terminal protein binding domain, another eliminates the C-terminal DNA binding domain entirely, and two appear to disrupt the structure in a small part of the C-terminal domain. These mutants will be useful for further studies of SeqA that measure the relative importance of the different domains to better understand the overall mechanisms and functions of the protein. iv Table of Contents: Title Page i Acknowledgements iii Abstract iv List of Figures and Tables vi Introduction 1 Materials and Methods 7 Media and Antibiotics 7 Bacterial Strains 7 Linker Scan method 11 PCR confirmation in pBAD18 12 Site Directed Mutagenesis 12 Megaprimer method 13 Electroporation Transformation 14 Survival Assays 15 Flow Cytometry 16 Antibody Purification 16 Protein-Protein Interaction Pull-down Experiment 17 Fluorescence Microscopy 18 SDS-PAGE and Western Blotting 19 SeqA Protein Purification 20 Results 22 Linker Scan Sequences 22 Sensitivity Assays 23 Flow Cytometry 29 Fluorescence Microscopy 31 Pull Down Assays 33 Discussion 34 Conclusion 36 References 37 v List of Figures and Tables: Figure 1: Crystal Structure of a SeqA dimer bound to hemimethylated DNA Table 1: Background Strains Table 2: Full Strain List Table 3: Primer List Figure 2: Linker Scan insertions in seqA Figure 3: DNA damage assay for Linker Scan mutants in seqA::FRT cat Figure 4: DNA damage assay for Linker Scan mutants in wildtype Figure 5: DNA damage assay for XL1-Red mutants in seqA::FRT cat Figure 6: DNA damage assay for Site Directed mutants in seqA::FRT cat Figure 7: Flow cytometric analysis of Linker Scan mutants in seqA::FRT cat Figure 8: Live Cell Fluorescence Microscopy images of Linker Scan mutants in seqA::FRT vi Introduction DNA replication is the most important process for evolutionary fitness because it passes the genetic material to the next generation. Most organisms copy their genome once and only once per cell cycle. This means that regulating replication is a critical function for an organism’s survivability. The prokaryote Escherichia coli is one of the few organisms that can replicate its genome multiple times in the same cell cycle. However, multiple rounds of replication must be carefully synchronized in order to ensure proper chromosome segregation for cell division. Once replicated, the new chromosomes must be separated to opposite ends of the cell, so that when the cell divides, each daughter cell has its own set of genetic material. When placed in nutrient rich media, E. coli replicates and divides approximately every twenty minutes. The rate at which DNA replication occurs depends on the frequency of replication initiation and the overall rate of DNA synthesis [1]. The timing of the eclipse period, or time between rounds of replication, is important in determining the timing of the entire cell cycle, and thus, the organism’s survival [2]. The eclipse period is approximately 60% of the generation time in wildtype strains [3]. Cell division, which has its own regulation mechanisms, must synchronize with replication. For this reason, generation time varies with the rate of replication initiation [3]. Replication begins at oriC, a gene that contains a large number of regulatory signal sequences and is approximately 245 base pairs. It includes an AT-rich region of 1 three 13-mer repeats and five binding sites for the replication initiator protein, DnaA [4]. These sites must be fully methylated for DnaA to have the highest affinity to its specific consensus sequence. In addition to these DnaA binding sites, oriC also contains 11 GATC sites in and around the DnaA binding sites, which serve as recognition sequences for DNA adenine methyltransferase (Dam) and SeqA [4, 5]. SeqA negatively regulates initiation by preventing DnaA from binding, while Dam positively regulates initiation by creating fully methylated GATC sites, to which SeqA cannot easily bind. Dam methylates adenine nucleotides in E. coli. E. coli uses Dam to distinguish its own DNA from foreign DNA [6]. Dam cannot bind if SeqA is already bound, both seeming to play antagonistic roles to each other with regard to regulating replication initiation. SeqA- deficient mutants have a similar phenotype to mutants that over express Dam [7]. Because SeqA has a significantly higher binding affinity to hemimethylated DNA than Dam [7], the two bind sequentially. This implies a potential binding partner of SeqA would remove it from DNA to allow Dam to bind. Initiation of replication happens when DnaA binds to oriC and recruits several replication factors to form the replisome, including DnaA-binding protein (DiaA). The newly formed DiaA and DnaA complex then begins to unwind the AT-rich region. The open region then recruits single stranded DNA-binding protein (SSB) [4]. Once oriC has opened up, two double hexamers of the helicase DnaB, which is bound to the helicase loader, DnaC, are loaded into the unwound section of DNA [8]. This second complex further unwinds the DNA and two replication forks begin replicating the DNA in a bidirectional method. 2 Replication initiation is regulated by several different factors. Because DnaA specifically recognizes methylated DNA, regulating the methylation of adenine nucleotides helps regulate the binding of DnaA. SeqA regulates the methylation process by competing with Dam at the 11 GATC sites to prevent Dam from methylating the adenine. Because of semi-conservative replication, GATC sites become hemimethylated, to which SeqA preferentially binds [9]. When SeqA binds these sites, reinitiation is inhibited because DnaA cannot bind the newly created DnaA boxes. Without SeqA, the eclipse period is reduced from 60% to 16% of its generation time [3]. The transcription of the dnaA gene is a second method of initiation regulation [9]. DnaA is located near oriC, and the SeqA binding of GATC sites near oriC also limits transcription of dnaA [9]. These factors regulate initiation differently depending on the cell’s environment. Nucleotide analogues, such as azidothymidine (AZT), and other DNA damaging agents, such as ultraviolet irradiation (UV), cause breaks in the DNA chain. This causes the replication fork to stall while the damage is being repaired [10]. Improper regulation of the bidirectional replication forks can lead to multiple unsynchronized forks to be present on the chromosome. A second replication fork can collide with a primary one, causing the forks to “fall off”. Without the reassembly of the replisome, the chromosome will be only partially replicated and it cannot properly segregate into the daughter cells, resulting in cells that are not viable. Because of this effect, seqA mutants show a higher sensitivity to AZT and UV induced damage [11]. SeqA also plays a necessary role in the stringent response by promoting replication arrest through initiation inhibition [12]. GATC sites are scattered throughout the E. coli genome, including several promoter regions that show SeqA-dependence [13]. More of these sites are found in 3 areas of low transcription and fewer in areas of high transcription [14]. This may correlate to SeqA binding, indicating that SeqA binding may have a role in gene regulation outside the replication origin [14]. SeqA binds to hemimethylated DNA, then forms a homodimer. These dimers can then bind to other SeqA dimers to form tetramers or multimers [15]. This, in turn, can mediate formation of aggregates, which indicate that SeqA foci track the replication forks [6]. Generally, GATC sites occur in clusters whose sites are 10, 19, 70, and 1100 base pairs apart, which SeqA strongly prefers [16]. This implies that SeqA binds two GATC sites that are approximately or two helix turns away from each other.
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