Biochemical Characterization Of The Rtcb Family Of Rna Ligases In Bacteria

Other Titles
Abstract
All known life relies on the proper transmission of genetic information through DNA and RNA to protein. The protection of this information is critical, and rejoining of broken DNA or RNA strands by ligases is essential for maintenance of genetic integrity. The intent of this thesis is to provide a broad survey of the strategies that Nature employs to rejoin broken polynucleotide strands, with an emphasis on RNA strand ligation, finally focusing on RtcB, which accomplishes RNA ligation by a novel mechanism. When RNA strands are broken, the fate of the phosphate at the break site dictates which enzymes must be employed to rejoin the broken ends. All ligases characterized before 2011 require a 5'-PO4 end and a 3'-OH end for ligation. In the instance that the products of the breakage are a 3'-PO4 or a 2',3'>PO4 and a 5'-OH, such as in the case of enzymatic transesterification by certain endonucleases, several enzymes are needed to reconcile these ends to a 5'-PO4 and 3'-OH before “classic” ligation may occur. This is known as the “healing and sealing” strategy. RtcB is able to bypass the need for healing by directly sealing a 2'3'>PO4 or 3'-PO4 end to a 5'-OH via a four-step mechanism in which (i) RtcB is covalently guanylylated at an active-site histidine, (ii) the guanylylated RtcB can hydrolyze a 2',3'>PO4, producing a 3'- PO4, (iii) RtcB transfers its GMP to the 3'-PO4, forming RNA3'pp5'G, and (iv) RtcB catalyzes nucleophilic attack of the 5'-OH on the phosphate atom of the 3'-PO4, sealing the RNA strand and releasing GMP. This thesis describes the structure-guided mutagenesis of the Escherichia coli RtcB active site. Separation-of-function mutants parsed the contributions of specific residues in specific steps of the overall ligation reaction and prove the reaction pathway that RtcB catalyzes. This thesis also describes characterization of three RtcB paralogs from Myxococcus xanthus, RtcB1, RtcB2, and RtcB3. The characterization of RtcB3 shows an enzyme that, despite extremely weak ligation activity, can guanylylate either a 5'- or 3'-PO4, and does so more efficiently on DNA than RNA.
Journal / Series
Volume & Issue
Description
Sponsorship
Date Issued
2016
Publisher
Keywords
DNA; Enzymology; Ligation; Repair; RNA
Location
Effective Date
Expiration Date
Sector
Employer
Union
Union Local
NAICS
Number of Workers
Committee Chair
Committee Co-Chair
Committee Member
Degree Discipline
Molecular Biology
Degree Name
Degree Level
Doctor of Philosophy
Related Version
Related DOI
Related To
Related Part
Based on Related Item
Has Other Format(s)
Part of Related Item
Related To
Related Publication(s)
Link(s) to Related Publication(s)
References
Link(s) to Reference(s)
Previously Published As
Government Document
ISBN
ISMN
ISSN
Other Identifiers
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International
Types
dissertation or thesis
Accessibility Feature
Accessibility Hazard
Accessibility Summary
Link(s) to Catalog Record