Browsing by Subject "Genome stability"

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Laboratory evolution and natural transformation in Acinetobacter baylyi ADP1
(2016-09-14) Renda, Brian Anthony; Barrick, Jeffrey E.; Bull, James; Ellington, Andrew; Alper, Hal; Davies, Bryan
The bacterium Acinetobacter baylyi ADP1 has been proposed as a next-generation chassis for genome engineering due to its high natural transformation rates and metabolic proficiency. Development of ADP1 for biotechnological use however is inhibited by the evolutionary instability of the competence, the large homology requirement for the efficient transformation of DNA into the ADP1 genome, and incomplete knowledge regarding the composition and regulation of the protein complex involved in DNA uptake. Chapters 1 and 2 describe an investigation of the evolutionary stability of competence. We conducted a 1000 generation evolution experiment with ADP1 and characterized the genetic basis of competence loss. We found that mutations caused by the mobile genetic element IS1236 mediated loss of were a driving force for genetic instability in the strain. We also found that a previously uncharacterized filamentous phage (CRAϕ) emerged from the ADP1 genome. CRAϕ appears to infect via the ADP1 competence machinery – providing a potential explanation for the fitness benefit of competence loss. Chapter 3 describes studies aimed at improving transformation rates and better characterizing the ADP1 transformation system. We examined how perturbing factors that affect the fate of internalized DNA effects transformation rates at different homology lengths. We found that some factors, such as exonuclease activity and recombinase activity, limited transformation rates at certain homology lengths and DNA concentrations. Other factors, such as adding random DNA to the ends of fragments to buffer against exonucleases and altering the abundance of a single-stranded DNA protecting protein, did not. Chapter 4 describes an effort to better characterize the machinery required for competence in ADP1. We coupled transformation-mediated selection of an ADP1 transposon library with Tn-seq to identify genes required for competence. Through this method, we were able to positively identify two additional competence genes (pilR and ACIAD3188) and other candidates. Together, the work presented in this dissertation provides insights into the evolutionary stability and molecular workings of ADP1 competence. These insights represent progress toward further improving ADP1 for use as a versatile genome and metabolic engineering platform.
Poly(ADP-ribose) polymerase-1 : protein substrates identification and biological functions characterization
(2018-05-02) Chen, Jung-Kuei; Liu, Hung-wen, 1952-; Whitman, Christian P.; Hoffman, David W.; Kim, Jonghwan; Miller, Kyle M.
PARP-1 catalyzes poly(ADP-ribosyl)ation reaction to mediate various cellular processes including DNA repair, chromatin remodelling, transcription, inflammation, and cell death. Despite its discovery for more than fifty years, many fundamental questions regarding the roles of PARP-1 in various DNA repair pathways remain unresolved. By comparing all available mass spectrometry-based screens to identify PARP-1 substrate proteins, cold-inducible RNA-binding protein (CIRBP) was commonly found to associate with PARP-1. Here I identify cold-inducible RNA-binding protein (CIRBP) as a newly identified key regulator in DNA double-strand break (DSB) repair. On DNA damage, CIRBP temporarily accumulates at the damaged regions and is poly(ADP ribosyl)ated by poly(ADP ribose) polymerase-1 (PARP-1). Its dissociation from the sites of damage may depend on its phosphorylation status as mediated by phosphatidylinositol 3-kinase-related kinases. In the absence of CIRBP, cells showed reduced γH2AX, Rad51, and 53BP1 foci formation. Moreover, CIRBP-depleted cells exhibited impaired homologous recombination, impaired nonhomologous endjoining, increased micronuclei formation, and higher sensitivity to gamma irradiation, demonstrating the active involvement of CIRBP in DSB repair. Furthermore, CIRBP depleted cells exhibited defects in DNA damage-induced chromatin association of the MRN complex (Mre11, Rad50, and NBS1) and ATM kinase. CIRBP depletion also reduced phosphorylation of a variety of ATM substrate proteins and thus impaired the DNA damage response. The role of PARP-1 in mediating DNA/RNA hybrid (R-loop) formation was also explored. Programmed R-loop formation was required to regulate diverse cellular processes, but uncontrolled R-loops accumulation is also a source of genome instability. The preliminary data suggest that PARP-1 interacts with several nucleolus proteins, and depletion of PARP-1 or these nucleolus proteins induces R-loop accumulation. Taken together, these results provide a better understanding of PARP-1 in DNA damage responses and genome integrity.