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

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

Bibliographic Details
Main Author: Smith, Christopher E.
Language:English
Published: The Ohio State University / OhioLINK 2015
Subjects:
Biochemistry
DNA repair
DNA recombination
recombineering
single strand annealing
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-osu14491833212021-08-03T06:34:22Z Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda; Smith, Christopher E. Biochemistry DNA repair DNA recombination recombineering single strand annealing 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. 2015 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321 http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321 unrestricted This thesis or dissertation is protected by copyright: some rights reserved. It is licensed for use under a Creative Commons license. Specific terms and permissions are available from this document's record in the OhioLINK ETD Center.
collection NDLTD
language English
sources NDLTD
topic Biochemistry
DNA repair
DNA recombination
recombineering
single strand annealing
spellingShingle Biochemistry
DNA repair
DNA recombination
recombineering
single strand annealing
Smith, Christopher E.
Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;
author Smith, Christopher E.
author_facet Smith, Christopher E.
author_sort Smith, Christopher E.
title Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;
title_short Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;
title_full Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;
title_fullStr Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;
title_full_unstemmed Insights into the structure and function of Red beta: the unique single-strand annealing protein of bacteriophage lambda;
title_sort insights into the structure and function of red beta: the unique single-strand annealing protein of bacteriophage lambda;
publisher The Ohio State University / OhioLINK
publishDate 2015
url http://rave.ohiolink.edu/etdc/view?acc_num=osu1449183321
work_keys_str_mv AT smithchristophere insightsintothestructureandfunctionofredbetatheuniquesinglestrandannealingproteinofbacteriophagelambda
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