PROTEIN/NUCLEIC-ACID STRUCTURE AND SEQUENCE REQUIREMENTS FOR SPECIFYING SECOND-STRAND CLEAVAGE AND SECOND-STRAND SYNTHESIS DURING THE INTEGRATION OF THE SITE-SPECIFIC LINE R2Bm

Author

Aruna Govindaraju

Graduation Semester and Year

2017

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Quantitative Biology

Department

Biology

First Advisor

Kathryn O'Donnell-Mendell

Second Advisor

Shawn Christensen

Abstract

Long interspersed nucleotide elements (LINEs) are major drivers of genomic landscaping events in many eukaryotic genomes. LINEs encode a multifunctional protein that binds to their own mRNA, recognize and cleave target DNA, and performs target-primed reverse transcription (TPRT). R2 element from Bombyx mori (R2Bm) has served as an important experimental system for the biochemical understanding of the structure, function, and integration of LINEs. First-strand target site cleavage and TPRT in the current model of integration have been well understood both in vitro (using R2Bm) and in vivo (using LINE-1 elements); however, interpretation of factors involved in second-strand cleavage and second- strand cDNA synthesis has been experimentally difficult. A 3D modeling of R2Bm endonuclease was performed in conjunction with in vitro studies of point mutants. The 3D model revealed that the endonuclease of R2, and R2-like elements consisted of a variant of PD-(D/E)XK motif that serves as the core catalytic motif for many restriction-like endonuclease-fold (RLE) family of nucleases. Point mutations revealed the position and function of the catalytic K in that motif and other DNA binding residues. The model is largely built based on the structure of archaeal Holliday junction resolvases and few restriction endonucleases. These findings invoked the investigation of R2 activity on branched DNA structures and the possibility of DNA structure playing a role in second-strand cleavage and synthesis. A preferential binding of R2 protein to a non-target four-way Holliday junction over a non-target linear DNA was shown. In addition, an efficient second-strand cleavage in a previously unrecognized four-way structure that mimics template switching event was achieved. Upon second-strand cleavage, the same intermediate could provide a template-primer for second-strand synthesis. Based on the findings of this dissertation work, a new updated model of LINE integration was proposed that emphasizes on both sequence and structural aspects of integration intermediates.

Keywords

LINE, TPRT, Long interspersed nucleotide elements, Target-primed reverse transcription

Disciplines

Biology | Life Sciences

License

This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

Comments

Degree granted by The University of Texas at Arlington

Recommended Citation

Govindaraju, Aruna, "PROTEIN/NUCLEIC-ACID STRUCTURE AND SEQUENCE REQUIREMENTS FOR SPECIFYING SECOND-STRAND CLEAVAGE AND SECOND-STRAND SYNTHESIS DURING THE INTEGRATION OF THE SITE-SPECIFIC LINE R2Bm" (2017). Biology Dissertations. 175.
https://mavmatrix.uta.edu/biology_dissertations/175