Structural and Functional Analysis of RecA-like Recombinases and Rationally-designed Peptides That Modulate the RecA Activities
Date Issued
2010
Date
2010
Author(s)
Chen, Li-Tzu
Abstract
This thesis focused on two subjects. One is the study on structure and function of Sulfolobus solfataricus (Sso) RadA. Another is the peptide (IRFLTARRR) derived from
structure of RecA-DNA complex can promote not only the enzymatic activity of RecA protein but also resistance to DNA damaging agents.
The RecA family of proteins is essential in homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against
DNA double strand breaks. In the previous reports, RecA family of proteins is thought to perform DNA strand exchange as a right-handed filament (active form) or as a
closed-ring (inactive form).
In this thesis, we report two new crystal structures that are left-handed and overwound right-handed helical filaments. Comparing the four different structures, we suppose that the DNA homology pairing and strand exchange occurs in the overwound right-handed nucleoprotein filament, and release of DNA exchange final products using the
left-handed filament. We also identified the conserved hinge region (subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. The results of several in vitro experiments are consistent with our hypothesis.
Another story is about a rationally-designed small peptide based on the Escherichia coli RecA-DNA crystal structure can promote homologous recombination through the enhancement of both RecA-mediated strand assimilation and three-strand exchange activity. We identified that the hydrophobicity and poly-positive charges, and the space between them in those small peptides are crucial features for such activities.
Remarkably, peptide #3 alone without RecA can also promote the D-loop formation at elevated temperature. Cell viability assays showed that the peptide elevates mammalian cell resistance to two cytotoxic DNA drugs, cisplatin and doxorubicin. The rescue of viability may result from increased DNA repair efficiency. Such peptides may find
future biological applications.
Subjects
RecA
homologous recombination
DNA repair
left-handed filaments
small peptides
drug design and strand exchange
Type
thesis
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