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

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

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. When it binds to

DNA, SeqA generates DNA supercoiling [17] and overwinds the DNA [18]. In the presence of topoisomerase I, SeqA has been shown to generate positive supercoiling in vitro and to change the twist or writhe of the DNA [17]. This supercoiling tightens DNA, inhibiting other proteins from binding and unwinding the DNA, which prevents both initiation of oriC and the promoter region of dnaA. The only other binding partner for

SeqA that has been found is ParC, one of the subunits of topoisomerase IV [16]; however, this interaction has yet to be studied in detail.

The structure of SeqA can provide clues as to the mechanisms of its various functions. The first 33 amino acids make up the N-terminal domain, which has been shown to bind other SeqA molecules, allowing it to dimerize [18]. The C-terminal domain, comprised of amino acids 65-181, is essential for binding the GATC sites. Some of the residues on the surface of the protein, such as R70, R73, E74, L77, and D79, form non-covalent bonds with residues on the surface of a mirroring SeqA dimer, forming tetramers [18]. The linker region in between, residues 34-64, make up the flexible hinge.

4 A majority of the residues in this region are hydrophobic and can form an amphipathic alpha-helix on the surface of the dimer [18]. Varying the length of the linker region can vary the ideal binding distance between GATC sites for the subunits of a SeqA dimer

[18]. For example, in the structure crystallized, residues 41-59 have been deleted and the

SeqA subunits bind GATC sites 9 base pairs apart rather than 10 [18]. By adding positively charged residues to the hinge region, SeqA becomes more prone to aggregation, indicating that this region might participate in forming multimers [19]. The two main regions are structurally distinct and each perform one of the essential SeqA functions, self-association with the N-terminal region and DNA binding with the C- terminal region, completely independently of the other domain [20].

Figure 1: Linear representation of the SeqA domains in the primary structure (top) and ribbon structure of a SeqA dimer bound to hemimethylated DNA [18] with the N-terminal SeqA binding domains in green and the C-terminal DNA binding domains in red (bottom).

5 Although the different functions of domains of SeqA have been fairly well characterized, the relative importance of those domains has not. By creating mutants that can disrupt SeqA-SeqA interactions but not DNA binding, or vice versa, the relative importance of the two functions can be determined. Many mutants of seqA were created, but only a handful yielded significant results. The most important mutants created were a large insertion in the N-terminal domain, which may disrupt SeqA-SeqA interaction, a deletion of the C-terminal domain, and two mutants that may have an effect on DNA binding. These mutants could prove interesting in future studies measuring the relative importance of dimerization and DNA binding in the function of SeqA.

Materials and Methods

Media and Antibiotics

Bacteria were primarily grown in rich Luria-Bertani (LB) media containing 1% w/v tryptone, 0.5% w/v yeast extract, and 0.5% w/v sodium chloride. LB plate media was the same as above with the addition of 1.5% w/v agar. Selection of strains was achieved by adding antibiotics to the final concentration of 100 µg/mL ampicillin (Ap),

15 µg/mL chloramphenicol (Cm), 30 µg/mL kanamycin (Kn), and/or 15 µg/mL tetracycline (Tc) when necessary. Plasmid gene regulation was achieved with 0.2% w/v glucose or arabinose.

Bacterial Strains

All bacterial strains used were Escherichia coli K-12 {isogenic to MG1655 [21]}.

Strains were constructed by electroporation transformation. Cell growth and culture density was determined by measuring the optical density at 600nm (O.D.600).

Table 1: Background strains Strain Genotype Source MG1655 (STL 242) F- rph-1 F.R. Blattner et al, (1997) [21] XL1-Blue (STL 284) F’ [proAB laclq lacZ∆M15 Stratagene ::Tn10] recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac DH5a (STL 2895) F- phi80d lacZ∆M15 Stratagene ∆(lacZYA-argF)U169 endA1 recA1 hsd17 deoR supE44 thi- 1 λ- gyrA96 relA1 STL 7222 seqA::FRT cat F- rph-1 CmR seqA gene disruption in MG1655 STL 7896 seqA::FRT F- rph-1 STL 7222 with cat gene flipped out

7 Table 2: List of Strains with Plasmids Strain Plasmid Plasmid Genotype Backgrou (STL number) nd 4083 pBAD18 amp XL1-Blue 10050 pBAD18-seqA amp seqA+ DH5a 11028 pBAD18 amp STL 7222

11032 pBAD18-seqA-Red1 amp seqA* STL 242 11033 pBAD18-seqA-Red2 amp seqA* STL 242 11034 pBAD18-seqA-Red3 amp seqA* STL 242 11035 pBAD18-seqA-Red4 amp seqA* STL 242 11072 pBAD18-seqA-Red5 amp seqA* STL 242 11289 pBAD18-seqA-Red7 amp seqA* STL 242 12843 pBAD18-seqA-Red9 amp seqA* STL 242 12844 pBAD18-seqA-Red10 amp seqA* STL 242 12845 pBAD18-seqA-Red11 amp seqA* STL 242 14715 pBAD18-seqA-Red1 amp seqA* DH5a 14716 pBAD18-seqA-Red2 amp seqA* DH5a 14717 pBAD18-seqA-Red3 amp seqA* DH5a 14718 pBAD18-seqA-Red4 amp seqA* DH5a 14719 pBAD18-seqA-Red5 amp seqA* DH5a 14720 pBAD18-seqA-Red7 amp seqA* DH5a 14721 pBAD18-seqA-Red9 amp seqA* DH5a 14722 pBAD18-seqA-Red10 amp seqA* DH5a 14723 pBAD18-seqA-Red11 amp seqA* DH5a 15369 pBAD18-seqA-Red1 amp seqA* STL 7222 15370 pBAD18-seqA-Red2 amp seqA* STL 7222 15371 pBAD18-seqA-Red3 amp seqA* STL 7222 15372 pBAD18-seqA-Red4 amp seqA* STL 7222 15373 pBAD18-seqA-Red5 amp seqA* STL 7222 15374 pBAD18-seqA-Red7 amp seqA* STL 7222 15405 pBAD18-seqA-Red9 amp seqA* STL 7222 15406 pBAD18-seqA-Red10 amp seqA* STL 7222 15494 pBAD18-seqA-Red11 amp seqA* STL 7222

12487 pBAD18-seqA-R30A amp seqA(∆76-91bp) STL 242 12488 pBAD18-seqA-R31A amp seqA(R31A) STL 242 12489 pBAD18-seqA-F106A amp seqA(315::+52bp) STL 242 12491 pBAD18-seqA-R118A amp seqA(R118A) STL 242 12493 pBAD18-seqA-W146A amp seqA(∆36-37bp,∆436bp) STL 242 14724 pBAD18-seqA-R30A amp seqA(∆76-91bp) DH5a 14725 pBAD18-seqA-R31A amp seqA(R31A) DH5a 14726 pBAD18-seqA-F106A amp seqA(315::+52bp) DH5a 14727 pBAD18-seqA-R118A amp seqA(R118A) DH5a 14728 pBAD18-seqA-W146A amp seqA(∆36-37bp,∆436bp) DH5a

8 15059 pBAD18-seqA-R30A amp seqA(∆76-91bp) STL 7222 15060 pBAD18-seqA-R31A amp seqA(R31A) STL 7222 15061 pBAD18-seqA-F106A amp seqA(315::+52bp) STL 7222 15062 pBAD18-seqA-R118A amp seqA(R118A) STL 7222 15063 pBAD18-seqA-W146A amp seqA(∆36-37bp,∆436bp) STL 7222

14266 pBAD18-seqA-LS1 amp seqA(533bp::Tn7∆cat) XL1-Blue 14269 pBAD18-seqA-LS2 amp seqA(54bp::Tn7∆kan) XL1-Blue 14550 pBAD18-seqA-LS3 amp seqA(219bp::Tn7∆cat) XL1-Blue 14551 pBAD18-seqA-LS4 amp seqA(481bp::Tn7∆cat) XL1-Blue 14552 pBAD18-seqA-LS5 amp seqA(196bp::Tn7∆cat) XL1-Blue 14565 pBAD18-seqA-LS6 amp seqA(479bp::Tn7∆cat) XL1-Blue 14731 pBAD18-seqA-LS1 amp seqA(533bp::Tn7∆cat) STL 242 14732 pBAD18-seqA-LS2 amp seqA(54bp::Tn7∆kan) STL 242 14733 pBAD18-seqA-LS3 amp seqA(219bp::Tn7∆cat) STL 242 14734 pBAD18-seqA-LS4 amp seqA(481bp::Tn7∆cat) STL 242 14735 pBAD18-seqA-LS5 amp seqA(196bp::Tn7∆cat) STL 242 14736 pBAD18-seqA-LS6 amp seqA(479bp::Tn7∆cat) STL 242 15052 pBAD18-seqA-LS1 amp seqA(533bp::Tn7∆cat) STL 7222 15053 pBAD18-seqA-LS2 amp seqA(54bp::Tn7∆kan) STL 7222 15054 pBAD18-seqA-LS3 amp seqA(219bp::Tn7∆cat) STL 7222 15055 pBAD18-seqA-LS4 amp seqA(481bp::Tn7∆cat) STL 7222 15056 pBAD18-seqA-LS5 amp seqA(196bp::Tn7∆cat) STL 7222 15057 pBAD18-seqA-LS6 amp seqA(479bp::Tn7∆cat) STL 7222

15824 pCA24N-His-seqA cat 6xHis seqA+ STL 7836 16057 pCA24N cat 6xHis STL 7836 15858 pCA24N-His-seqA-LS2 cat 6xHis XL1-Blue seqA(54bp::Tn7∆kan) 15859 pCA24N-His-seqA-LS3 cat 6xHis XL1-Blue seqA(219bp::Tn7∆cat) 15860 pCA24N-His-seqA-LS4 cat 6xHis XL1-Blue seqA(481bp::Tn7∆cat) 15861 pCA24N-His-seqA-LS6 cat 6xHis XL1-Blue seqA(479bp::Tn7∆cat) 15862 pCA24N-His-seqA-Red3 cat 6xHis seqA* XL1-Blue 15863 pCA24N-His-seqA-R30A cat 6xHis seqA(∆76-91bp) XL1-Blue 15864 pCA24N-His-seqA-F106A cat 6xHis seqA(315::+52bp) XL1-Blue 15865 pCA24N-His-seqA-W146A cat 6xHis seqA(∆36- XL1-Blue 37bp,∆436bp) 15888 pCA24N-His-seqA-LS2 cat 6xHis STL 7836 seqA(54bp::Tn7∆kan) 15889 pCA24N-His-seqA-LS3 cat 6xHis STL 7836 seqA(219bp::Tn7∆cat)

9 15890 pCA24N-His-seqA-LS4 cat 6xHis STL 7836 seqA(481bp::Tn7∆cat) 15891 pCA24N-His-seqA-LS6 cat 6xHis STL 7836 seqA(479bp::Tn7∆cat) 15892 pCA24N-His-seqA-Red3 cat 6xHis seqA* STL 7836 15893 pCA24N-His-seqA-R30A cat 6xHis seqA(∆76-91bp) STL 7836 15894 pCA24N-His-seqA-F106A cat 6xHis seqA(315::+52bp) STL 7836 15895 pCA24N-His-seqA-W146A cat 6xHis seqA(∆36- STL 7836 37bp,∆436bp)

16181 pGAP40-GFP-seqA amp GFP seqA+ STL 7836 16182 pGAP40-GFP-seqA-LS2 amp GFP DH5a seqA(54bp::Tn7∆kan) 16183 pGAP40-GFP-seqA-LS3 amp GFP DH5a seqA(219bp::Tn7∆cat) 16184 pGAP40-GFP-seqA-LS4 amp GFP DH5a seqA(481bp::Tn7∆cat) 16185 pGAP40-GFP-seqA-LS6 amp GFP DH5a seqA(479bp::Tn7∆cat) 15161 pET104.1-BBD-seqA amp BBD seqA+ DH5a 16262 pET104.1-BBD-seqA amp BBD seqA+ STL 7836 16263 pGAP40-GFP-seqA-LS2 amp GFP STL 7836 seqA(54bp::Tn7∆kan) 16264 pGAP40-GFP-seqA-LS3 amp GFP STL 7836 seqA(219bp::Tn7∆cat) 16265 pGAP40-GFP-seqA-LS4 amp GFP STL 7836 seqA(481bp::Tn7∆cat) 16266 pGAP40-GFP-seqA-LS6 amp GFP STL 7836 seqA(479bp::Tn7∆cat)

13392 Linker Scan pool amp seqA** XL1-Blue 14235 pBAD18-seqA-LS1 amp seqA(533bp::Tn7) XL1-Blue (full transposon) 14237 pBAD18-seqA-LS2 amp seqA(54bp::Tn7) XL1-Blue (full transposon) 14444 pBAD18-seqA-LS3 amp seqA(219bp::Tn7) XL1-Blue (full transposon) 14445 pBAD18-seqA-LS4 amp seqA(481bp::Tn7) XL1-Blue (full transposon) 14450 pBAD18-seqA-LS5 amp seqA(196bp::Tn7) XL1-Blue (full transposon) 14442 pBAD18-seqA-LS6 amp seqA(479bp::Tn7) XL1-Blue (full transposon)

14374 pwsk29 amp STL 7222 (plasmid from STL 1285) 14375 pwsk29-pDEST14-seqA amp seqA+ STL 7222 (plasmid from STL 7864) 14376 pwsk29-pDEST14-seqA amp seqA+ STL 7222 (plasmid from STL 7865) 14377 pDEST14-seqA amp seqA+ STL 7222 (plasmid from STL 7867) 14378 pGAP40-GFP-seqA amp GFP seqA+ STL 7222 (plasmid from STL 8122) *indicates unknown mutation **indicates mixed population of mutants

Linker Scan method

Linker scan mutants were created using New England BioLabs (NEB) GPS™

Linker scanning kit and Epicentre Linker scanning kit by the protocol in the manual provided with the kit. First, 2µL 10X GPS buffer and 1µL donor DNA from the kit was added to 0.08µg target DNA and dH2O to a total volume of 18µL. Then, 1µL of

Transposase was then added, and the mixture was incubated in a 37ºC water bath for 10 minutes. Finally, 1µL start solution was then added and the mixture was incubated in a

37ºC water bath for 1 hour. This mixture was then transformed into XL1-Blue cells and plated on the appropriate selective media.

Colonies were then patch streaked and underwent colony PCR (polymerase chain reaction). The PCR product was run on a 0.8% agarose gel and compared with a 1kb standard ladder. Products on the gel that show the linker scan insertions in seqA were then frozen away from the cells on the corresponding patch streak. These cells also had the plasmid extracted using a Sigma GenElute™ plasmid kit, then the plasmid underwent a restriction digest, using PmeI (NEB kit) and NotI (Epicentre kit) from NEB, under the optimal conditions listed in the NEB restriction digest manual. The plasmid was then

11 religated and sent to Dana Farber Cancer Institute for sequencing and frozen and stored at

-80ºC.

PCR for confirmation pBAD

Potential Linker Scan mutants were screened using colony PCR. After undergoing the linker scan reaction (described above), a single colony was picked with a sterile toothpick and resuspended in 15µL sterile ddH2O and microwaved for 30 seconds at the highest power. This was then used as the template DNA in the PCR reaction and was mixed with 12.5µL GoTaq® PCR Master Mix from Promega.

Primers for sequencing and PCR on the plasmid pBAD18 were

5’-CTGTTTCTCCATACCCGTT-3’ for the forward direction and

5’-CTCATCCGCCAAAACAG-3’ for the reverse direction [22] and created by Sigma.

A volume of 0.5µL of each primer (resuspended to a 10µM solution) was added to the reaction mixture.

PCR reactions were performed in a MJ Research Peltier Thermal Cycler PTC-200 using the following schedule:

1) 95ºC for 2 min, 25 times [2) 95ºC for 30s, 3) 50ºC for 30s, increase by 1ºC per cycle up to 55ºC, 4) 72ºC for 3 min 30s], 5) 72ºC for 5 min, 6) 4ºC indefinitely

Site Directed Mutagenesis

Site directed mutants were created by Vincent Sutera using outward PCR [23] with the following primers:

12 Table 3: Primers used for Site Directed Mutagenesis of SeqA Mutant Forward Primer Reverse Primer R30A 5’-CGTATGTTGAAATTTTCC 5’-CGCTAAAATGTCGGAT GCCGCATCACAG-3’ GCGCTCTCGCCGAT-3' R31A 5’-ATGTTGAAATTTTCCGCC 5’-AGCCCGTAAAATGTCG GCATCACAGCCT-3’ GATGCGCTCTCGCC-3’ F106A 5’-GCCGAAGCAACGGAATC 5’-AGCCGCCTGGGCGTCA GTTGCACGGTCGT-3’ AGAGAATATAGTGT-3’ R118A 5’-GTTTACTTTGCGGCAGAT 5’-GGCTGTACGACCGTGC GAACAAACGCTG-3’ AACGATTCCGTTGC-3’ W146A 5’-GTGATCACCAACACCAA 5’-CGCATACGGCGTGCCT CACCGGCCGTAAA-3’ GGCACATGTTTCGG-3’

Megaprimer method

To clone mutants from one vector to another, the mutants underwent PCR as described above, but using the following primers that anneal to seqA:

5’-GCATTGAAGTTGATGATGAACTCTAC-3’ for the forward primer, and

5’-GATAGTTCCGCAAACCTTCTCAATC-3’ for the reverse primer. The product was then purified using a Qiagen QIAquick® PCR purification kit. This purified product then serves as both primers for a second PCR, with the new vector (with seqA+) as the template. A volume of 1µL of these primers was added to 10µL H5 reaction buffer, 1µL dNTP mix, 2.5µL template DNA, 0.5µL Phusion, and 35µL ddH2O. The following schedule proved most efficient:

1) 95ºC for 2 min, 24 times [2) 95º for 45s, 3) 72ºC for 6 min], 4) 95ºC for 45s, 5)

72ºC for 16 min, 6) 4ºC indefinitely

The PCR product then underwent a restriction digest using DpnI to eliminate the template DNA as described in the NEB restriction digest manual. The new plasmid was then transformed into XL1-Blue and frozen for long-term storage.

13 Electroporation Transformation

Electroporation transformation was completed in order to introduce plasmids carrying desired mutations into specific strain backgrounds.

Competent cells of STL242 (MG1655), STL284 (XL1-Blue), STL2895 (DH5a),

STL7222, and STL7836 were made by the following protocol. A standing overnight of culture was grown in 10mL LB media at 37ºC using selective media when appropriate.

A larger culture was then inoculated using the overnight culture in a 1:100 dilution and grown with vigorous shaking at 37ºC to an OD600 of 0.6-0.8. The culture was then centrifuged at 4,000 rpm for 10 minutes at 4ºC and the supernatant decanted. Cells were resuspended in approximately 200mL cold 1mM Hepes, pH 7.0. Cells were centrifuged again at 4,000 rpm for 10 minutes at 4ºC and the supernatant decanted. Cells were resuspended in ~100mL cold 7% dimethyl sulfoxide (DMSO). A final centrifugation was done for 10 minutes at 4,000 rpm at 4ºC and resuspended in 3mL cold 7% DMSO.

Cells were stored at -70ºC.

Transformations were performed using a BioRad GenePulser™. First, 1mL LB media was aliquoted into sterile test tubes. When necessary, DNA was drop dialyzed to remove salt using 0.025µm Millipore nitrocellulose filters. Then, 40µL competent cells were added to a control test tube. Then, 40µL competent cells were mixed with 2-3µL plasmid DNA in a sterile microcentrifuge tube and then transferred to an electroporation cuvette and pulsed. The 1mL of LB media was then added to the cuvette to resuspend the cells using a sterile pipetteman tip. The resuspended cells were then poured back into the test tube and placed in a rollerdrum at 37ºC for 1 hour. 100-200µL of each culture was

14 placed onto selective plate media and incubated overnight at 37ºC. Growth was expected on test plates, but not on the control plate.

Survival Assays (AZT + UV)

Viability of strains when subjected to DNA damaging agents was assessed through azidothymidine (AZT, Sigma) and ultraviolet irradiation (UV) survival assays according to the following protocol. Single colonies were put in 1mL selective LB media with glucose. Cultures were grown while shaking at 37ºC for 2 hours. Cultures were then transferred to microcentrifuge tubes and centrifuged at 13,000g for 1 minute. The supernatant was discarded and the pellet was resuspended in 300µL LB media. 150µL culture was then added to each 1mL selective LB media with glucose and 1mL selective

LB media with arabinose. Cultures were grown while shaking at 37ºC for 2 hours. Then,

200µL of each culture was pipetted into a 96-well plate and 1:10 serial dilutions through

10-5 into 56/2 salt solution were completed in the adjacent wells, each addition was followed by adequate resuspension. Following dilution, 5µL of each well was plated on eight LB plates and two each of the following doses of AZT in LB plate media: 25 ng/mL, 50 ng/mL, 75 ng/mL, and 100 ng/mL. Two LB plates at a time were placed in a transluminator exposed to UV radiation at the following concentrations: 20 J/m2, 40 J/m2, and 60 J/m2. Two LB plates served as the base line for both AZT and UV exposure.

Each plate also had the same sugar (glucose or arabinose) in which those samples were originally grown. The plates were then incubated overnight at 37ºC. Colonies were then counted for each strain assayed and fraction survival was calculated at each given AZT and UV dose.

15 Flow Cytometry

DNA content and replication synchrony were assessed using flow cytometry. A

2mL standing overnight cultures of strains were grown at 37ºC in LB media with glucose and appropriate antibiotics. The following day, cultures were diluted 1:100 into fresh media of either glucose or arabinose and grown with vigorous shaking at 37ºC to an

OD600 of 0.2. At this point, 1mL of culture was fixed in 9mL of 95% ethanol. The remaining culture was treated to a final concentration of 300 µg/mL rifampicin and 32

µg/mL cephalexin. The culture was grown for an additional 2-3 hours to complete already initiated replication events. An additional 1mL of culture was then fixed in 9mL of 95% ethanol.

Samples were spun down at 4,000 rpm for 10 min at 4ºC and resuspended in

500µL phosphate-buffered saline (PBS). Invitrogen Pico Green dye was diluted 1:100 in

25% DMSO and 100µL was added to each sample. The samples then sat in total darkness for 1-3 hours at room temperature, then diluted using 500µL of a 1:1000 dilution of Pico Green dye in 1x PBS. Samples were analyzed using an Accuri® C6

Flow Cytometer and its accompanying software CFlow® Version 1.0.202.1.

Antibody Purification

SeqA antibody came from a Rabbit Anti-serum from Yenzym, which was purified using the subtraction method. A standing overnight of STL7836 was diluted 1:100 in LB media and grown with vigorous shaking at 37ºC to an OD600 of 0.6-0.8. The culture was then centrifuged at 4,000 rpm for 10 minutes at 4ºC and the supernatant decanted. The cells were then resuspended in 2xFSB without Bromophenol Blue {2.5mL Tris (pH=6.8),

4.0mL 10% sodium dodecyl sulfate (SDS), 2.0mL Glycerol, 1.0mL ß-mercaptoethanol

16 (BME), and 0.5mL ddH2O}. This mixture was then put on top of a 6.5cm x 9cm piece of nitrocellulose paper and allowed to incubate, while gently shaking, at 4ºC for 2 hours.

The cell suspension was removed, then 5mL of the anti-serum was added and allowed to incubate, while gently shaking, on the nitrocellulose for 2 hours. The serum was then pipetted out into microcentrifuge tubes and frozen at -20ºC.

Protein-Protein Interaction Pull-down Experiment

His-tagged and Green Fluorescent Protein fusion (GFP) constructs of seqA+ were expressed using the Lac promoter on the vectors pCA24N and pGAP40, respectively. A standing overnight of culture was grown in 5mL LB media at 37ºC using selective media when appropriate. A larger culture was then inoculated using the overnight culture in a

1:100 dilution and grown with vigorous shaking at 37ºC to an OD600 of 0.6-0.8.

Isopropyl ß-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of

1mM, and the culture was allowed to grow for an additional 2 hours. The culture was then centrifuged at 4,000 rpm for 10 minutes at 4ºC and the supernatant decanted. The cells were then resuspended in 1/100 of the original volume in Tris-Sucrose {50mM Tris

(pH=8.0), 10% Sucrose}.

Crude lysates of over-expressed cells were extracted by thawing 100µL of cells and adding 4µL of 25mM EDTA, 1µL of 1mM dithiothreitol (DTT), and 4µL of 10 mg/mL lysozyme in Tris-Sucrose to the cells in a microcentrifuge tube. This mixture was incubated on ice for 5 minutes, then 200mM of NaCl was added and incubated for an additional 25 minutes. The cells were then heat shocked three times for 15 seconds each in a 37ºC water bath, then placed on ice for 15 seconds in between each heat shock. They were then centrifuged at 13,000 rpm for 15 minutes in a microcentrifuge at 4ºC. A

17 sample of crude lysate was taken for SDS-PAGE, along with a sample of whole cells

(before lysis).

The pull-down experiments were completed using Nickel beads (Qiagen) with the following protocol: 100µL of a 50% slurry of Ni-NTA agarose beads were prepared with

3 washes of 500µL blank lysis buffer (BLB) {1mM DTT, 200mM NaCl, 10% ultrapure sucrose, 5mM Tris (pH=7.5)}. All centrifugations of the beads were done at 4,900 rpm and all other manipulations were done at 4ºC. After each wash, the beads were centrifuged for 1 minute and the supernatant was discarded. After preparation, equal volumes of beads and crude lysate of His-tagged SeqA (SeqA-His) or linker scan mutant were mixed and rotated for one hour. Another 10µL sample was taken for SDS-PAGE, then the beads were washed 3 times with 500µL wash buffer {50mM Tris (pH=8.0),

150mM NaCl}. After each wash, the beads were centrifuged for one minute and the supernatant was pipetted off and discarded. The beads were then resuspended in 100µL wash buffer, and a 10µL sample was taken for SDS-PAGE.

While SeqA-His mixed with the beads, SeqA-GFP was lysed using the same procedure detailed above. Equal volumes of SeqA-GFP crude lysate and SeqA-His mixed nickel beads were mixed and rotated for 1 hour at 4ºC. A 10µL sample was taken, then the beads were washed 3 times in the same manner as above. Finally, a 20µL sample was taken and SDS-PAGE was performed on the samples.

Fluorescence Microscopy

Standing overnight cultures were diluted 1:100 into 2mL fresh LB media in amber test tubes. Cultures were grown shaking at 37ºC for 1 hour. 1mL was taken out and put in a fresh amber test tube along with 250µL 4’,6-diamidino-2-phenylindole (DAPI) for a

18 working concentration of 2 µg/mL. Cultures were allowed to incubate shaking at 37ºC for 10-15 minutes. Samples were then centrifuged at 13,000 rpm for 1 minute in a microcentrifuge. The supernatant was decanted, and the pellet was then resuspended in

1mL fresh LB media. 10µL was spotted onto microscope slides with pads of 2% agarose.

Images were taken using an Olympus BX51 microscope equipped with a RGB liquid crystal color filter, and compiled using Velocity® v5.4.2.

SDS-PAGE and Western Blotting

Crude lysates and protein products from the pull-down assay were mixed with equal volumes of 2x FSB {4% sodium dodecyl sulfate (SDS), 200mM DTT, 120mM Tris

(pH 6.8), 0.002% bromophenol blue, 10% glycerol} and stored at -20ºC until ready for

SDS-PAGE. Frozen samples were thawed and equal volumes were loaded onto a 15%

SDS-PAGE gel with a 4% stacking gel according to the procedure found in BioRad Mini

Protean II Manual.

One gel of each set was stained using Coomassie Brilliant Blue {0.25%

Coomassie Brilliant Blue R250, 45% methanol, 10% glacial acetic acid, 45% dH2O} for

15 minutes. The gel was then washed three times in high destain solution {45% methanol, 10% glacial acetic acid, 45% dH2O} for 15 minutes each, then left indefinitely in low destain solution {10% methanol, 5% glacial acetic acid, 85% dH2O}.

Gels were transferred onto a PVDF membrane following the procedure described in Mini

Trans-Blot® Electrophoretic Transfer Cell Instruction Manual provided by BioRad®.

Membranes were blotted following the protocol described QIAexpress® Detection and

Assay Handbook, Protocol 7, using a primary Qiagen Penta-His antibody and secondary anti-mouse IgG antibody (HRP) from GE Healthcare for the His-tag, and the purified

19 Yenzym SeqA antibody (see above) with secondary anti-rabbit antibody (HRP) from GE

Healthcare for the SeqA peptide. For GFP detection, an anti-GFP IgG HRP conjugated antibody from Invitrogen was used. The same protocol was used for this antibody, except the incubation in primary antibody and subsequent wash steps were skipped.

Membranes were then treated using the SuperSignal® West Pico Chemiluminescent detection system manufactured by Thermo Scientific.

SeqA Protein Purification

His-tagged proteins were expressed as described above. A column of nickel beads was used to purify the His-tagged proteins. 2mL Ni-NTA Agarose (Qiagen) was prepared by adding 20 column volumes of Wash Buffer {50mM sodium phosphate

(pH=8.0), 500mM NaCl, 20mM Imidazole}. Cells were lysed as described above.

Samples of whole cells and crude lysate were set aside for SDS-PAGE. Crude lysate was then added to the column and allowed to gravity drain through the column. Flow-through was collected in a sterile microcentrifuge tube. 20 columns of Wash Buffer were then allowed to pass through the column and were collected by a separate, sterile container.

6mL of Elution Buffer {50mM sodium phosphate (pH=8.0), 500mM NaCl, 500mM

Imidazole} was added to the column and allowed to pass through. Six sterile, microcentrifuge tubes, were used to collect 1mL of elution each. Samples from all the elutions, along with whole cells, crude lysate, flow through, and wash steps, were mixed with equal volumes of 2xFSB and run on two SDS-PAGE gels as described above. One gel was stained with Coomassie and the other was probed with a His-tag antibody using the Western Blot protocol described above.

20 Protein concentration was determined using BioRad Protein Assay dye, diluted

1:5 in ddH2O. 2µL crude lysate, 2µL flow-through, 25µL wash, and 15µL of each elution were each added to 1mL of the diluted dye. Samples were analyzed in a Hitachi

U-2910 Spectrophotometer at an absorbance wavelength of 595nm. Absorbance for each of the nine samples was recorded and compared to a standard curve to determine protein concentration.

Results

Figure 2: Graphical representation of the locations of the Linker Scan insertions in seqA

Linker Scan sequences

LS1: The first Linker Scan mutant, labeled LS1 above, has a 15 base pair insertion between nucleotides G533 and C534, resulting in Val-Stop codons immediately following residue C178. This replaces the last three residues with a single Valine.

LS2: The second Linker Scan mutant, labeled LS2 above, has a 57 base pair insertion between nucleotides T54 and A55, resulting in a 16 residue insertion plus a three amino acid duplication (K19, H20, I21) on either side. The peptide sequence now reads (N’)-T-K-H-I-L-S-L-V-H-I-L-R-P-Q-D-V-Y-K-R-Q-K-H-I-G-(C’) instead of (N’)-

T-K-H-I-G-(C’).

LS3: The third Linker Scan mutant, labeled LS3 above, has a 15 base pair insertion between nucleotides T219 and G220, resulting in Val-Stop codons immediately

22 after residue L75. This eliminates more than half of the SeqA protein, including nearly the entire DNA binding domain.

LS4: The fourth Linker Scan mutant, labeled LS4 above, has a 15 base pair insertion between nucleotides G481 and A482, resulting in the following peptide sequence inserted immediately after residue I160: (N’)-V-F-K-Q-I-(C’).

LS5: The fifth Linker Scan mutant, labeled LS5 above, has a 15 base pair insertion between nucleotides A196 and A197, resulting in the following peptide sequence inserted immediately after residue K66: (N’)-D-M-F-K-Q-(C’).

LS6: The sixth Linker Scan mutant, labeled LS6 above, has a 15 base pair insertion between nucleotides T479 and C480, resulting in Val-Stop codons immediately following residue I160. This replaces the last 20 amino acids with a single Valine residue.

Sensitivity Assays

The strain seqA::FRT cat with an empty pBAD18 vector consistently shows a strong sensitivity to both AZT and UV damage [11]. When the empty vector is replaced by pBAD18-seqA+ under the arabinose promoter, this sensitivity phenotype is rescued in the presence of arabinose. LS2, LS3, LS4, and LS6 show sensitivity to UV radiation, and

LS3 and LS6 show sensitivity to AZT (Figure 3). LS2 shows only a partial rescue in

AZT sensitivity. Only LS1 and LS5 show a rescued phenotype when induced. All of the linker scan mutants show a similar sensitivity to the empty vector when plated uninduced.

These plasmids were then put into a wildtype background to see if the mutations are dominant or recessive. All of the linker scan mutants have the same sensitivity as the wildtype version of SeqA, indicating none of the mutants are dominant (Figure 4).

Figure 3: DNA sensitivity assays of Linker Scan mutants in seqA::FRT cat background. The top graphs show relative survivability to increasing UV radiation of the mutants uninduced (left) and induced (right). The bottom graphs show the same for AZT exposure.

Figure 4: DNA sensitivity assays of Linker Scan mutants in wildtype background. The top graphs show relative survivability to increasing UV radiation of the mutants uninduced (left) and induced (right). The bottom graphs show the same for AZT exposure.

25 The same damage assays were completed to test the mutants created by Nathan

Kaplan using XL1-Red mutagenic strain (Figure 5) and site directed mutants created by

Vincent Sutera (Figure 6). Mutant Red3 showed a null phenotype, but all the other XL1-

Red mutants showed wildtype phenotypes in the assays. However, when this mutant was later sequenced, it appeared to have a wildtype sequence. The site directed mutants,

R30A, F106A, R118A, and W146A all showed similar sensitivities as the negative control, while R31A showed no change in phenotype. When later sequenced, R30A,

F106A, and W146A all showed additional insertions and/or deletions. R30A has 16 nucleotides deleted, from T76 to C91, which results in a frameshift mutation for a majority of the gene. F106A has a 52 nucleotide insertion between G315 and T318, with

T316 and T317 apparently deleted, which also results in a frameshift mutation beginning in the middle of the DNA binding domain. W146A has T36 and A37 deleted, along with

T436, which results in a frameshift mutation between these two deletions. This mutation would alter more than half of SeqA, including most of the residues that have been shown to have an effect on the function.

Figure 5: DNA sensitivity assays of XL1-Red mutants in seqA::FRT cat background. The top graphs show relative survivability to increasing UV radiation of the mutants uninduced (left) and induced (right). The bottom graphs show the same for AZT exposure.

Figure 6: DNA sensitivity assays of Site Directed mutants in seqA::FRT cat background. The top graphs show relative survivability to increasing UV radiation of the mutants uninduced (left) and induced (right). The bottom graphs show the same for AZT exposure.

28 Flow Cytometry

Flow cytometry was used to analyze the four Linker Scan mutants that showed increased sensitivity to AZT and UV: LS2, LS3, LS4, and LS6 (Figure 7). Readings were taken on samples from both before and after the addition of replication inhibitors rifampicin and cephalexin to determine DNA content and synchrony of replication. None of the mutants showed sharp peaks under run-out conditions, indicating that DNA replication was not well synchronized, as it would be with functioning SeqA. This confirms that the Linker Scan mutants do not complement seqA::FRT cat, as also shown in the sensitivity assays.

Figure 7: Flow cytometric analysis of DNA content in a seqA::FRT cat background after two hours incubation with 300 µg/mL rifampicin and 32 µg/mL cephalexin (final concentrations). The labeled mutants and controls are all on the vector pBAD18, and samples are being induced with arabinose.

30 Fluorescence Microscopy

The same four Linker Scan mutants; LS2, LS3, LS4, and LS6; were cloned into pGAP40-seqA+-GFP using the megaprimer method and viewed as live cells under a confocal microscope (Figure 8). Wildtype SeqA-GFP forms distinct foci around DNA.

LS2 appears to be bound to DNA all over the cell, but does not form foci. This indicates a weakening of SeqA-SeqA interactions. LS3 and LS4 appear to have diffuse GFP spread throughout the cell, indicating that they do not bind DNA or form foci. LS6 seems to form a few foci at one end of the cell, but does not appear to be bound to DNA around the cell. a) b) c)

d) e) f)

Figure 8: Live cell imaging of GFP-fusions and DAPI stain in seqA::FRT background. a-c) wildtype SeqA with DAPI filter, GFP filter, and merge, respectively. d-f) seqA::FRT with no plasmid with DAPI filter, GFP filter, and merge, respectively.

31 g) h) i)

j) k) l)

m) n) o)

p) q) r)

Figure 8 (cont.): Live cell imaging of GFP-fusions and DAPI stain in seqA::FRT background. g-i) Mutant LS2 with DAPI filter, GFP filter, and merge, respectively. j-l) Mutant LS3 with DAPI filter, GFP filter, and merge, respectively. m-o) Mutant LS4 with DAPI filter, GFP filter, and merge, respectively. p-r) Mutant LS6 with DAPI filter, GFP filter, and merge, respectively.

32 Pull Down Assays

Pull Down experiments were attempted with biotinylated SeqA on Streptavidin beads, but the SeqA-BBD construct did not express properly. The same experiment was attempted with His-tagged SeqA on nickel beads, and a SeqA-GFP construct was tested for interactions. The SeqA-GFP was expressed in its entirety as shown on the SDS-

PAGE gel (data not shown), but the pull down was unsuccessful. The N-terminal GFP construct may have blocked interactions with the N-terminal domain of SeqA, causing the protein not to be pulled down; however, the same construct still formed foci in the live cell imaging, indicating the potential for interaction. Due to these setbacks, relative protein-protein binding of the mutants to wildtype SeqA could not be obtained.

Discussion

Although not essential, seqA provides an important level of control over the replication cycle. Linker Scan mutants have been created that change each part of SeqA: one that extends the N-terminal domain to interrupt its normal function, one that extends the linker region by five amino acids, one that eliminates the DNA binding domain entirely, and two that disrupt the last helix, which may disrupt the structure of the domain.

Each of these mutants affects the function of SeqA in different ways. LS1 changes only the last few amino acids, which has no apparent effect on SeqA function.

This means that the C-terminal residues have little to no effect on the function or structure of SeqA. LS2, the large insertion in the N-terminal domain, shows increased sensitivity to DNA damage relative to the wildtype protein, but decreased sensitivity relative to seqA::FRT cat mutants, which is indicative of a hypomorph. Because this mutation is in the N-terminal domain, the phenotype is probably caused by disruptions in the SeqA-SeqA interactions. This is also supported by fluorescence microscopy, where

GFP appears to localize around DNA without forming clear foci. LS3 adds a stop codon around the 75th amino acid, which deletes most of the DNA binding domain and eliminates the primary function of SeqA. This is why it shows as much sensitivity to

DNA damaging agents as the null strain. The crystal structure of SeqA shows that the two domains have few interactions with each other (see Figure 1) [18], indicating that this

34 mutant could potentially be used as a control when measuring SeqA-SeqA binding because it has an intact N-terminal domain, assuming that domain still folds properly.

Although LS5 shows a wildtype phenotype in DNA damage sensitivity, it lengthens the linker region. This may increase the flexibility between the two domains or increase the optimal binding distance between hemimethylated GATC. This mutant may prove useful in the future for experiments testing the function of the linker region, along with a mutant that shortens this region, such as the one used to obtain the crystal structure [13].

LS4 and LS6 have similar effects on SeqA itself, but somewhat different phenotypes. LS4, which inserts five amino acids after I160, only appears to show sensitivity to UV radiation and not AZT. LS6, which deletes all the amino acids after

I160, is sensitive to both UV and AZT damage. As seen in the fluorescence microscopy,

LS6 appears to have some ability to form foci, while LS4 does not. The addition of five amino acids at this location might disrupt the DNA binding domain by dislocating some of the secondary structures, whereas deleting these amino acids does not have an effect on the rest of the domain.

Conclusion

This investigation has yielded several new mutants of SeqA that can be used to characterize the protein further. The mutant LS2 appears to disrupt the N-terminal domain while LS3 eliminates the C-terminal domain, and LS 4 and LS6 appear to disrupt the C-terminal domain. The phenotypes of these mutants under DNA damaging conditions have been explored, but the specific molecular causes of these phenotypes warrant further study. The different mutants can serve as controls for each other when looking at the effects of one domain.

Further studies would explore the biochemical functionality of the individual domains and how they contribute to interactions between SeqA and DNA. Pull down assays and DNA binding assays could determine the relative importance of the two domains of SeqA in its sensitivity to DNA damage. More detailed fluorescence microscopy could be used to determine the localization of mutated SeqA around DNA.

These mutants can ultimately be used to better understand SeqA, and its precise role in

DNA replication.

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