Browsing by Author "Kreuzer, Kenneth N"
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Item Open Access A Genetic Screen for the Identification of Mutants Hypersensitive to 5-Azacytidine(2010) Wu, Sunny YangA DNA-protein crosslink is a covalent bond between DNA and a protein. It is a type of DNA damage that is relatively understudied. This study reports on the identification of a set of transposon mutants sensitive to 5-azacytidine, a DNA- protein crosslink induction agent that induces a crosslink between DNA and a DNA methyltransferase protein. The screen showed that certain recombination, DNA repair, and tRNA modification mutants are hypersensitive to 5-azacytidine. These included the recombination recA, recC, and recG mutants. Since the recombination mutants consistently show high sensitivity to aza-C, it suggests a role for recombination in DNA-protein crosslink repair. Western blots for the levels of methyltransferase protein showed that mutants have similar levels of methyltransferase protein compared to wild type cells, arguing that the mutants' hypersensitivity to aza-C is not because of increased methyltransferase levels. Western blots for the levels of SsrA tagging in the presence of 5-azacytidine showed that the tRNA modification transposon mutants miaA, mnmE, and mnmG are all defective in SsrA tagging, which likely explains their hypersensitivity. The SsrA tag Western blots also unexpectedly showed that the recA transposon mutant had reduced levels of SsrA tagging when treated with 5-azacytidine.
Item Open Access A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle.(2012) Yong, Sheila T.Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis, a form of programmed cell death, has been extensively studied. However, since the developmental defects observed in Bat3‐null mouse embryos cannot be explained solely by defects in apoptosis, I investigated whether BAT3 is also involved in regulating cell‐cycle progression. Using a stable‐inducible Bat3‐knockdown cellular system, I demonstrated that reduced BAT3 protein level causes a delay in both the G1/S transition and G2/M progression. Concurrent with these changes in cell‐cycle progression, I observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21. p21 is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Additionally, I observed that the p21 turnover rate was also reduced in Bat3‐knockdown cells released from G2/M synchronization. My findings indicate that in Bat3‐knockdown cells, p21 continues to be synthesized during cell‐cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent cell‐cycle delay. Finally, I showed that BAT3 co‐localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. My study reveals a novel, non‐apoptoticrole for BAT3 in cell‐cycle regulation. By maintaining low p21 protein level during G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 into S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation, an event that promotes G2/M progression. BAT3 modulates these pro‐ and anti‐proliferative roles of p21 at least in part by regulating the translocation of p21 between the cytoplasm and nucleus of the cells to ensure proper functioning and regulation of p21 in the appropriate intracellular compartments during different cell‐cycle phases.Item Open Access DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase and Quinolone Antibiotics in Escherichia coli(2014) Henderson, MorganExpression of mutant M.EcoRII protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after aza-C incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork protection by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by inducing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep and ftsK as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.
Item Open Access Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.(2011) Bateman, Stacey LynnEscherichia coli is a typical constituent of the enteric tract in many animals, including humans. However, specialized extraintestinal pathogenic E. colistrains (ExPEC) may transition from benign occupation of the enteric and vaginal tracts to sterile sites such as the urinary tract, bloodstream, and central nervous system. ExPEC isolates of urinary tract origin express type 1 pili as a critical virulence determinant mediating adherence to and invasion into urinary tract tissues. Type 1 pili expression is under epigenetic regulation by a family of site-specific recombinases, including FimX, which is encoded from a genomic islet called PAI-X for Pathogenicity Islet of FimX. A goal of this study was to determine the prevalence of the type 1 pili epigenetic regulator genes (fimB, fimE, fimX, ipuA, ipuB) and associated PAI-X genes (hyxR, hyxA, hyxB) present among an extended, diverse collection of pathogenic and commensal E. coli isolates. Using a new multiplex PCR, fimX and the additional PAI-X genes were found to be highly associated with ExPEC (83.2%) and more prevalent in ExPEC of lower urinary tract origin (87.5%) than upper urinary tract origin (73.6%) or human commensal isolates (20.6%; p < 0.05, all comparisons). Fim-like recombinase genes ipuA and ipuB also had a significant association with ExPEC compared to commensal isolates, but had a low overall prevalence (23.8% vs. 11.1%; p < 0.05). PAI-X also showed a strong positive correlation with the presence of virulence genes in the genomes of pathogenic isolates. Combined, our molecular epidemiology studies indicate PAI-X is highly associated with ExPEC isolates, and its high prevalence suggests a potential role in the ExPEC lifestyle. Further investigation into the regulation of PAI-X factors showed that FimX is also an epigenetic regulator of a LuxR-like response regulator HyxR, encoded on PAI-X. In multiple clinical ExPEC isolates, FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the inversion sites of the type 1 pili promoter and independent of integration host factor IHF. Additional studies into the role of HyxR during ExPEC pathogenesis uncovered that HyxR is involved in regulation of the nitrosative stress response. In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNI), primarily through derepression of hmpA, encoding a nitric oxide detoxifying flavohemoglobin. However, in the macrophage, HyxR expression produced large effects on intracellular survival in the presence and absence of RNI, and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX. ExPEC reside in the enteric tract as commensal reservoirs, but can transition to a pathogenic state by invading normally sterile niches, establishing infection, and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. This study demonstrates the functional versatility of the FimX recombinase and identifies novel epigenetic and transcriptional regulatory controls for ExPEC tolerance to RNI challenge and survival during intracellular macrophage infection. Further investigation of these pathways may shed light on the regulatory cues and programming that provoke the commensal to pathogen transition.Item Open Access Fate of Transcription Elongation Complexes Stalled by DNA Damage and Elongation Inhibitors(2014) Krasich, RachelTranscription is the process by which cells translate genetic information stored in DNA into RNA. Transcription is a highly regulated and discontinuous process, and elongation is frequently blocked by DNA damage, pause sites, or intrinsic or external inhibitors. Due to the essential nature of transcription, the cell has numerous ways of dealing with these blockages to transcription, only some of which are understood. We examined the fate of RNA polymerase stalled by DNA-protein crosslinks as well as elongation inhibitors Streptolydigin and Actinomycin D.
We use 5-azacytidine, a cytosine analog that covalently traps cytosine methyltransferases, as a model system for DNA-protein crosslinks (DPCs) inEscherichia coli. Our lab previously showed the importance of the tmRNA system for survival during DPC-formation, implying that transcription and translation are blocked by DPCs. For tmRNA to function, the A-site must be cleared, requiring either RNA polymerase to be released first or the nascent RNA chain to be cleaved. Using cell growth assays, we tested mutants related to A-site cleavage factors known to affect transcription initiation, elongation, and termination. Of these mutants, only DksA seemed to have a mild effect, and only at late stages of growth phase. However, western blots for tmRNA tagging showed that dksA mutants have increased rather than decreased tmRNA tagging, indicating that another unknown factor is responsible for enabling tmRNA activity.
Since the issue of repair of DPCs remains unresolved, and the repair of DPCs could affect the blocked elongation complex, we used the same cell growth assay to look for potential repair pathways. We found that dnaK knockouts were slightly resistant to 5-azacytidine treatment which, coupled with our previous finding that dnaJ mutants are hypersensitive to DPCs, implies a potential DnaK-independent role for DnaJ in DPC repair.
Previous in vitro studies have shown that Stl-stalled RNAP is stable, while in vivo studies argued that Stl-inhibited polymerases are released from the DNA transcript, implying that there is a release factor responsible for removing RNAP from DNA in vivo. Using cell growth assays, Western blots for tmRNA tagging, and in vitro studies, we showed the transcription-coupled repair factor Mfd is responsible for releasing Stl-stalled RNAP, and that treatment with an elongation inhibitor such as Stl is an effective treatment against cells overexpressing the transcription-coupled repair pathway.
The tmRNA western blots also implied that Mfd has termination abilities in wildtype cells, leading us to perform RNAseq analysis on mfd knockout and overexpressing cells. We found that global transcription patterns are changed by altering Mfd levels, thus allowing us to propose a novel transcription regulatory role for Mfd.
We extended our elongation inhibitor studies to the eukaryotic inhibitor Actinomycin D and found that transcription-coupled repair pathway is again involved in responding to stalled RNAP. We also screened rifampicin-resistant RNAP mutants for Actinomycin D resistance and found several with the desired phenotype. We thus propose that Actinomycin D inhibition is more complicated than just steric hindrance due to DNA intercalation.
Item Open Access In Vivo Analysis of the Consequences and the Repair Mechanisms of Azacytidine-Induced DNA-Protein Crosslinks(2009) Kuo, Hung-Chieh Kenny5-azacytidine and its derivatives are cytidine analogs used for leukemia chemotherapy. The primary effect of 5-azacytidine is the prohibition of cytosine methylation, which results in covalent DNA-methyltransferase crosslinks at cytosine methylation sites. These DNA-protein crosslinks have been suggested to cause chromosomal rearrangements and contribute to cytotoxicity, but the detailed mechanisms of DNA damage and the repair pathways of DNA-protein crosslinks have not been elucidated.
We used 2-dimensional agarose gel electrophoresis and electron microscopy to analyze plasmid pBR322 replication dynamics in Escherichia coli cells grown in the presence of 5-azacytidine. 2-dimensional gel analysis revealed the accumulation of specific bubble- and Y-molecules, dependent on overproduction of the cytosine methyltransferase EcoRII and treatment with 5-azacytidine. Furthermore, a point mutation that eliminates a particular EcoRII methylation site resulted in disappearance of the corresponding bubble- and Y-molecules. These results imply that 5-azacytidine-induced DNA-protein crosslinks block DNA replication in vivo. RecA-dependent X-structures were also observed after 5-azacytidine treatment. These molecules may be generated from blocked forks by recombinational repair and/or replication fork regression. In addition, electron microscopy analysis revealed both bubbles and rolling circles after 5-azacytidine treatment. These results suggest that replication can switch from theta to rolling circle mode after a replication fork is stalled by a DNA-methyltransferase crosslink. The simplest model for the conversion of theta to rolling-circle mode is that the blocked replication fork is cleaved by a branch-specific endonuclease. Such replication-dependent DNA breaks may represent an important pathway that contributes to genome rearrangement and/or cytotoxicity.
In addition, we performed a transposon mutagenesis screen and found that mutants defective in the tmRNA translational quality control system are hypersensitive to 5-azacytidine. The hypersensitivity of these mutants requires expression of active methyltransferase, indicating that hypersensitivity is dependent on DNA-methyltransferase crosslink formation. Furthermore, the tmRNA pathway is activated upon 5-azacytidine treatment in cells expressing methyltransferase, resulting in increased SsrA tagging of cellular proteins. These results support a "chain-reaction" model, in which transcription complexes blocked by 5-azacytidine-induced DNA-protein crosslinks result in ribosomes stalling on the attached nascent transcripts, and the tmRNA pathway is invoked for cleaning up the resulting pile-ups. In support of this model, an ssrA mutant is also hypersensitive to antibiotic streptolydigin, which blocks RNA polymerase elongation. These results reveal a novel role for the tmRNA system in clearance of coupled transcription/translation complexes in which RNA polymerase has become blocked.
Item Open Access Involvement of a DNA Polymerase III Subunit in the Bacterial Response to Quinolones(2014) Whatley, Zakiya NicoleQuinolone treatment induces stabilized cleavage complexes (SCCs), consisting of a covalent gyrase-DNA complex, and processing of these complexes is thought to cause double-strand breaks and chromosome fragmentation. SCCs are required but not sufficient for cytotoxicity; the mechanism that converts SCCs to double-strand breaks is not clearly understood. Evidence of chromosome fragmentation due to quinolones comes from indirect measures such as sedimentation analysis of nucleoids and measurements of lysis viscosity. This work outlines a method that combines agarose plugs, conditional lysis and field inversion gel electrophoresis to allow direct visualization of chromosomal fragmentation resulting from quinolone treatment. We are able to distinguish between latent breaks within the stabilized cleavage complex and irreversible breaks that result from downstream processing.
When seeking to understand the genetic requirements for quinolone-induced SOS response, we found that a dnaQ mutant has a specific defect in SOS induction following nalidixic acid. The product of dnaQ is the ε subunit of DNA polymerase III, which provides 3' → 5' exonuclease activity. In addition to the nalidixic acid-specific SOS defect, δdnaQ has multiple phenotypes: slow growth, high mutation frequency, and constitutive SOS. We propose that ε has a role in the quinolone response beyond the normal proofreading function of the subunit in the polymerase III core. Using a unique transposon mutagenesis system, we created a library of dnaQ mutants with 15 base pair insertions that were scored phenotypically. We identified mutants that separated the various phenotypes, arguing strongly that ε has multiple functions. The isolation of a stable dnaQ mutant with SOS phenotypes allows the study of this function without confounding results from spurious mutations throughout the chromosome. We also isolated a novel class of SOS "hyper-inducible" mutants. Additionally, my findings with weak and strong β-clamp binding mutants provides the first in vivo characterization of these ε mutants and gives insight into the SOS response following nalidixic acid treatment.
Item Open Access Native Origins for Constitutive Stable DNA Replication in Escherichia Coli(2012) Maduike, Nkabuije ZikaodiConstitutive stable DNA replication (cSDR) is an alternative mode of replication initiation in Escherichia coli. cSDR is active in rnhA and recG mutants, which lack proteins that remove DNA-RNA hybrids called R-loops. The mechanism for cSDR initiation, therefore, is thought to involve these R-loop structures, which are proposed to form at specific locations known as oriK sites on the chromosome. Thus far, oriK sites have only been mapped to broad, 100-200 kb regions on the chromosome, so the specific elements involved in initiation are still unknown. My research focused on localizing the oriK sites on the chromosome, specifically those in the terminus region, where two of the major oriK sites had previously been mapped. We used two-dimensional gel electrophoresis (Friedman & Brewer, 1995) to analyze the replication forks that are blocked at the innermost Ter sites at the terminus, and found that elevated levels of replication forks are blocked at the Ter sites in rnhA mutants. We also used microarray and deep sequencing analysis to determine that there is a major location of oriK activity in the chromosome, located in the region between TerA and TerC. Furthermore, we also studied the use of the activation-induced deaminase (AID) enzyme as a tool for identifying regions of R-loop formation in the chromosome, and learned about its properties in the process.
Item Open Access Purification and Characterization of Novel Denitrosylases from Yeast and Mammals(2012) Anand, PuneetS-nitrosylation, the prototypic mechanism of redox-based signal transduction, involves the covalent attachment of a nitrogen monoxide group to a Cys-thiol side chain. S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions and protein stability. The primary focus of my dissertation was to solve a problem of great importance in the field of S-nitrosylation, which is, to identify denitrosylase(s) i.e., enzymes that remove NO groups from S-nitrosothiols. Recent progress in elucidating the cellular regulation of S-nitrosylation has led to the identification of two physiologically relevant denitrosylating activities that remove the NO group from S-nitrosylated substrates. Thioredoxin/thioredoxin reductase (Trx system) functions as an NADPH-dependent denitrosylase across a broad range of S-nitrosylated proteins (SNO-proteins). S-nitroso-glutathione reductase (GSNOR), which is highly conserved across phylogeny, metabolizes GSNO utilizing NADH as a reducing coenzyme, thereby shifting equilibria between GSNO and SNO-proteins. This dissertation describes the discovery of two novel denitrosylases: one from yeast and the other from mammals. Using technique of column chromatography we have purified these novel denitrosylases to homogeneity and have demonstrated a principal contribution of these enzymes towards S-nitrosothiol metabolism.
Item Open Access The Development and Testing of a System for Monitoring Site-Specific Lesions In Vivo(2013) Asllani, MelissaEvery day, cells face agents that generate lesions in genomic DNA, which can interfere with the processes of DNA replication and gene expression. These lesions can range from small abasic sites to alkylated bases to large proteins frozen on the DNA and can be caused by both endogenous and exogenous agents. These lesions must be repaired to maintain genomic stability, and multiple pathways exist to perform the necessary repairs or to bypass the damage. These pathways have been discovered and studied using a variety of experimental techniques, both in vitro and in vivo. While these studies have contributed valuable information about many of cellular processes, there are still gaps in the DNA repair field.
The goal of this study is to bridge some of those gaps by constructing a system to introduce DNA containing a site-specific lesion into Escherichia coli cells at high enough levels to monitor the lesion's fate in vivo and in real time. This system combines two separate DNA molecules to simplify the introduction of a site-specific lesion. The first molecule is the DNA from bacteriophage λ, a virus that is able to infect E. coli cells at a high level of efficiency. A typical commercial packaging reaction can yield titers of approximately 1.0 x 109 plaque forming units (PFU)/mL. However, bacteriophage λ has a large genome of approximately 48.5 kb, which makes it a difficult substrate for extensive cloning and manipulation. In contrast, cloning and manipulation of a small plasmid (~4 kb) is a much simpler endeavor, and small plasmids have been used previously to produce DNA containing a site-specific lesion. The problem with using a plasmid occurs when attempting to introduce it into cells, as the process of transformation is not very efficient and can cause unintended consequences in the cells. This new system allows for the incorporation of the lesion into the plasmid, which is then integrated into a bacteriophage λ vector, λ Kytos. The combination of these two molecules produces bacteriophage λ DNA containing a site-specific lesion, which can infect the cells at high efficiency, allowing the fate of the DNA to be monitored in real-time. Interestingly, repair of a single EthenoA lesion after infection appears to be a very inefficient process. Even if the repair system is activated by induction with methyl methanesulfonate (MMS) or if individual repair proteins are overexpressed, little to no repair occurs. As methylation occurs upon injection, it does appear that the DNA is exposed to proteins in the cell, including any repair proteins present. These results indicate that other processes, perhaps replication or transcription, are required to repair a single EthenoA lesion in vivo.
Item Open Access The Study of Fork Processing and DSB Repair in Bacteriophage T4: Roles of the MR Complex and Endonuclease VII(2013) Almond, Joshua RichardAbstract
During DNA replication, organisms often encounter different types of DNA damage or lesions that lead to the stalling or breakage of replication forks. Cells must repair this damage or it can lead to genome instability and the loss of cell viability. It is now accepted that recombination plays a vital role in the rescue and restart of replication forks. The study of replication fork processing by recombination is important for understanding how cells avoid DNA damage which, if unrepaired, can lead to cancer or cell death.
One of the most detrimental forms of DNA damage experienced by the cell is a double-stranded DNA break (DSB). DSBs can be repaired in an error-free manner by homologous recombination through many different proposed mechanisms. Homologous recombination is of particular interest as defects in the recombination proteins often lead to deadly human syndromes characterized by a predisposition to cancer. For example, mutations in the BRCA2 protein, which is involved in homologous recombination for the repair of DSBs, have been linked to an increased incidence of breast cancer. Therefore, our lab is interested in studying the mechanism of homologous recombination and in particular how it relates to stalled fork processing and DSB repair.
We studied the different characteristics of DNA recombination using bacteriophage T4 as a model system. Phage T4 is a well-characterized virus that infects Escherichia coli. Phage T4 has a simple genome that encodes for almost all of the proteins involved in its own replication, recombination, and repair. At late times of infection, phage T4 predominantly uses recombination-dependent replication (RDR) to replicate its DNA. This recombination-dependent replication system is very similar to homologous recombination used by higher organisms. One major advantage of using phage T4 as a model system is that unlike other model systems, "lethal" mutations can be examined in T4 using strains that suppress specific mutations. Using this system, we investigated a few of the phage T4 proteins (homologous with higher organism proteins) that are thought to be involved in DSB repair and replication fork processing and whose roles are not yet fully defined.
One such protein that we studied was gene product 47 (gp47). The gp47 protein is a member of the bacteriophage T4 Mre11/Rad50 complex and is homologous to the human Mre11 protein (gp47 will be referred to as T4 Mre11). We investigated the in vivo functions of T4 Mre11 in double-strand end processing, double-strand break repair, and recombination-dependent replication. Specifically, we tested the importance of the conserved histidine residue in nuclease Motif I (catalytic active site) of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. Our collaborator, Dr. Scott Nelson, also constructed and expressed a T4 Mre11 mutant protein with the same conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind T4 Rad50 and double-stranded DNA. These results indicate that the nuclease activity of T4 Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.
Another such protein that we studied was gene product 46 (gp46). The gp46 protein is the other member of the bacteriophage T4 Mre11/Rad50 complex and is homologous to the human Rad50 protein (gp46 will be referred to as T4 Rad50). We investigated the in vivo functions of T4 Rad50 in double-strand end processing, double-strand break repair, and recombination-dependent replication. Specifically, we tested different aspects of the protein such as the Signature motif, which is part of the nucleotide binding domain (ATPase active site). Substitution with different ATPase-deficient mutations completely blocked plasmid recombination-dependent replication and led to the stabilization of double-strand ends. These results indicate that the ATPase activity of T4 Rad50 is critical for recombination-dependent replication during T4 infections.
Finally, we investigated Endonuclease VII (gp49), which is encoded by gene 49. EndoVII was the first endonuclease shown to cleave Holliday junctions in vivo and was shown to cleave stalled replication forks in vitro. We were interested in investigating the proposed regulation of EndoVII expression along with EndoVII's role in replication fork processing. Specifically, we used the EndoVII hairpin mutation to address the proposed regulation of EndoVII expression. In the presence of WT EndoVII we observed only the full-length EndoVII protein, whereas in the presence of the EndoVII hairpin mutant, we observed no EndoVII protein. This result indicates that EndoVII is not regulated to express different length proteins at different times of infection.
We also used the EndoVII hairpin mutation to investigate EndoVII's role in replication fork processing. We generated phage strains to monitor EndoVII's effect on recombination using a phage x phage recombination assay. We observed no difference in recombination between WT and HP- strains. Taken with the above conclusions, we propose that the EndoVII hairpin mutation disrupts the late promoter and reduces the amount of active EndoVII available. In turn, this would affect the phage's ability to package its DNA due to unresolved recombination junctions and its ability to survive.