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Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;

Smith, Christopher E
http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Bacteriophage lambda encodes a two-component Synaptase-Exonuclease (SynExo) system used for generating end-to-end concatemers of the viral genome before packaging. Lambda exonuclease (exo) is a processive 5'-3' exonuclease that degrades linear dsDNA into mononucleotides, generating a 3' single-stranded overhang. Redß is a single-strand annealing protein (SSAP) that binds to the resulting overhang and anneals it to a complementary strand. Redß serves as a model to study the conserved single-strand annealing (SSA) DNA repair pathway, yet its manner of oligomerization, DNA binding and annealing remain elusive due to difficulty in obtaining a high-resolution structure. Here, we quantitatively compare the characteristics of the N-terminal DNA binding domain of Redß (Redß177) with that of the full-length protein by measuring their DNA binding, in vivo activity, and interaction with exo. RedßFL and Redß177 have unique DNA binding preferences to ssDNA and annealed duplex product, and display significant differences in recombination activity, revealing that the C-terminus of Redß is required for full in vivo activity and a robust interaction with its intrinsic partner, exo. The C-terminal residues 182-261 (RedßCT) fold into a monomeric, alpha-helical structure and are sufficient in forming an interaction with exo, yet are unable to bind ssDNA or annealed duplex. Additionally, we show the ability of Redß177 to form oligomeric structures similar to those of RedßFL, alone and in the presence of DNA. Redß oligomerization is a highly dynamic and concentration-dependent process, and the presence of DNA substrates stabilizes the oligomer. The Redß-exo synaptosome complex also appears to be highly dynamic, and we propose a unique model for Redß-exo processing of dsDNA ends. As we continue to pursue a 3D crystal structure for Redß, data presented here supports a modular domain structure for Redß: an N-terminal domain that contains the determinants for ssDNA binding and oligomerization; and a C-terminal domain that is required for in vivo activity and contains the determinants for synaptosome formation with exo.
Charles Bell (Advisor)
180 p.
Biochemistry
DNA repair; DNA recombination; recombineering; single strand annealing

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Citations

  • Smith, C. E. (2015). Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda; [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321

    APA Style (7th edition)

  • Smith, Christopher. Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321.

    MLA Style (8th edition)

  • Smith, Christopher. "Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321

    Chicago Manual of Style (17th edition)

osu1449183321
576
© 2015, some rights reserved.Creative Commons License Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda; by Christopher E Smith is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.
Release 3.2.12