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Chi Subunit of Polymerase III Holoenzyme May Have Function in Addition to Facilitating DNA Replication

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Chi Subunit of Polymerase III Holoenzyme May Have Function in Addition to Facilitating DNA Replication Chi Subunit of Polymerase III Holoenzyme May Have Function in Addition to Facilitating DNA Replication Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biochemistry Dr. Susan Lovett, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Biochemistry by Taku Harada May 2018 Copyright by Taku Harada © 2018 Acknowledgments I would like to thank my advisor Dr. Susan Lovett. Thank you for sharing this opportunity to explore the E. coli genome with you. I had a wonderful experience. Along the way, your unwavering support and encouragement was irreplaceable. I am greatly fortunate and appreciative. Thank you to my mentor Dr. Alex Ferazzoli. No amount of words could ever describe the gratitude I have for you. You were a mentor for me in science and life. Thank you for always supporting and encouraging me to learn even if, at times, it meant failure. I attribute my success to your investment and confidence in me. Most importantly, your enthusiasm and joyous personality is inspirational and made every day a good day. Thank you to Ariana, Dr. Cooper, Laura, Vinny, Julie, McKay and everyone who worked in the Lovett lab during my stay. You all welcomed me in and provided a supportive environment that extended beyond the lab walls. I am very fortunate to have worked with all of you. Thank you to all my friends. Special thanks Adib, Eli, Jessie, and Rich. My four years at Brandeis have been phenomenal because of your support and motivation. I look forward to many more years of friendship. Thank you to all the teachers at the Science, Math, and Technology Center at Godwin. Thank you for allowing me to explore and build a solid foundation to become the scientist I am today. Thank you to my family for always encouraging me to reach for the stars and providing me with the opportunities and support to do so. Your love and care for me is always appreciated. iii ABSTRACT Chi Subunit of Polymerase III Holoenzyme May Have Function in Addition to Facilitating DNA Replication A thesis presented to the Department of Biochemistry Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Taku Harada The chi subunit, encoded by the holC gene, is known to have a significant role in the efficient replication of DNA. It is unknown whether chi has other functions in the cell and it was the goal of this study to elucidate interaction of chi with other proteins. I screened suppressor mutations for a ΔholC Escherichia coli strain to determine what genes may be linked to chi function. Suppression of ΔholC mutants was done by culturing the strain in unrestricted growth and replicative stress conditions by exposure to AZT. Colonies that were larger in size relative to others were identified as suppressors and measured for viability and sensitivity to AZT. A total of 79 suppressor colonies were isolated. Many suppressors had greater viability and resistance to AZT relative to ΔholC mutants. Furthermore, suppressors had morphological changes including elongation of the cell and development of a mucoid membrane. These observations were more pronounced in suppressors isolated from AZT exposure. Twenty of these suppressors were whole genome sequenced and mutations in dnaB, sspA, rpoA, and rpoC were found. These mutations indicated previously unknown interactions with holC. I propose that the chi subunit may have additional functions to its established role in DNA replication, particularly DNA transcription. iv Table of Contents List of Tables ………………………………………………………………………………………………………………………………………. VI List of Figures …………………………………………………………………………………………………………………………………..... VII Introduction .................................................................................................................................................. 1 DNA Replication and holC ......................................................................................................................... 1 DNA Repair and holC ................................................................................................................................. 8 Materials and Methods .............................................................................................................................. 10 Bacterial strains and growth conditions ................................................................................................. 11 Isolation and identification of suppressor mutations ............................................................................. 12 Phenotypic measurements: Viability, AZT sensitivity, Microscopy ........................................................ 12 DNA extraction and Next-generation whole genome sequencing ......................................................... 13 Data analysis ........................................................................................................................................... 13 Results......................................................................................................................................................... 14 Initial screen for ΔholC suppressor mutations ........................................................................................ 14 ΔholC suppression and its control .......................................................................................................... 15 Large scale screen for ΔholC suppressor mutations ............................................................................... 18 Discussion ................................................................................................................................................... 25 References .................................................................................................................................................. 30 v List of Tables Table 1: Escherichia coli K-12 strains used/made in this study 10 Table 2: PCR and sequencing primers used in study 14 Table 3: ΔholC mutant suppressor strains from initial screen 15 Table 4: Phenotypes of suppressor colonies 23 Table 5: Phenotypes of sequenced suppressors 23 Table 6: ΔholC mutant suppressor strains from large scale screen 24 vi List of Figures Figure 1: Schematic of Replisome 2 Figure 2: Schematic of Pol III HE and its interaction with SSB 2 Figure 3: The gamma clamp loader complex hydrolyzes ATP to load the beta clamp 6 Figure 4: Chi subunit of gamma complex displaces primase from SSB to initiate replication 8 Figure 5: Azidothymidine is a chain terminator 9 Figure 6: ΔholC suppression controlled by changing growth conditions 17 Figure 7: tdk gene screen isolated suppressors with large deletions in the gene 19 Figure 8: Suppressor colonies grew robustly and were more resistant to AZT than ΔholC 20 Figure 9: Suppressor colonies have auxotrophic phenotype 21 Figure 10: Suppressor colonies have mucoid coating 23 vii Introduction DNA Replication and holC Chromosomal replication is an essential process for organisms to pass genetic information down from one generation to the next. This replication process is performed by the replisome which is present in all organisms with highly conserved structural and functional components (reviewed in Yao, 2010). The replisome of Escherichia coli has proven to serve as an excellent model for understanding each component’s function. When the replication process is unstable, changes in the genome can occur. These changes are known as mutations and can lead to catastrophic outcomes such as cell death. For this reason, it is important to understand how organisms can adapt to limit these occurrences. I am interested in replication stalling events and their effects on mutation. The replisome is an apparatus of multiple protein subunits responsible for the duplication of genomic DNA (Figure 1). It includes DnaB helicase, DnaG primase and the DNA Polymerase III holoenzyme (DNA Pol III HE) (Yao, 2010). The DNA Pol III HE synthesizes new DNA in Escherichia coli (Figure 2). DNA Pol III HE is composed of three polymerase cores (Pol III core) (McInemy, 2007), a beta clamp, and the gamma complex otherwise known as the clamp loader (Onrust, 1995). Each Pol III core is responsible for hydrolyzing ATP to rapidly add nucleotides and elongate DNA (McInemy, 2007). It is stabilized to the replication fork by binding to the beta clamp (Stukenberg, 1991). The beta clamp is essential for the synthesis of DNA and is “loaded” to encircle duplex DNA by the gamma complex (Stukenberg, 1991). The gamma complex recognizes primed DNA templates and hydrolyzes ATP to initiate this loading process (Simonetta, 2009). It is made up of five subunits. They are: gamma, delta, delta prime, chi, psi (Onrust, 1993). Of these subunits, only the gamma, delta, and delta prime subunits have been deemed essential for 1 the E. coli genome replication (Onrust, 1993). However, strains lacking chi and psi show major growth defects highlighting their importance in efficient genome duplication. 2 Replication initiates when DnaA opens the double-stranded DNA (dsDNA) to two single- strands (ssDNA), the leading and lagging strand. There is one origin of replication that creates two replication forks proceeding in a bidirectional manner. Single-stranded binding protein (SSB) binds to the ssDNA and then recruits components of the replisome (Yao, 2010). DnaB helicase unwinds the dsDNA further (Biswas, 1999). The DnaG primase synthesizes RNA primers onto both the leading and lagging single-stranded DNA (ssDNA)
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