TelA Promoted Telomere Resolution Features an Underwound Pre-cleavage Intermediate
Date
2023-06-13
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Masters
Abstract
Agrobacterium tumefaciens and Borrelia species are two examples of prokaryotic organisms that have linear replicons, in contrast to most prokaryotes that have circular genomes. These organisms can replicate the lagging strand end of linear (deoxyribonucleic acid) DNAs without the loss of the DNA’s information due to the covalently closed DNA hairpin telomeres at the termini of the DNA. Replication of DNAs with hairpin telomeres produces a circular inverted repeat dimer with replicated telomere junctions. This intermediate cannot be segregated into two daughter cells without a two-step DNA breakage and rejoining process known as telomere resolution. Specialized enzymes known as telomere resolvases create hairpin telomeres from a dimeric replication intermediate through telomere resolution. Telomere resolvases share a similar mechanism to that of topoisomerase-IB and tyrosine recombinase enzymes. The proposed models for telomere resolution include a pre-cleavage intermediate where the base pairing between the scissile phosphates is broken helping to propel the reaction forwards. A class of variants was examined in previous studies on the borrelial telomere resolvase, ResT. These variants were inactive on parental substrates but were rescued by substrate alterations that mimicked DNA unwinding between the cleavage sites. In ResT, the catalytic domain and the hairpin-binding module cooperate to stabilize an underwound pre-cleavage intermediate. The idea that the telomere resolvase from Agrobacterium tumefaciens, TelA, and ResT follow the same reaction pathway was generated from the crystal-structure data of TelA, and the homology between ResT and TelA. We generated TelA variants homologous to those in ResT that stabilize the pre-cleavage intermediate. We also generated TelA variants of sidechains that were shown to interact with the hairpin turnarounds in TelA structures. Reaction analysis showed that the central two basepairs of the replicated telomere (rTel) junction are likely to be underwound and stabilized by TelA in a pre-cleavage intermediate. We also have evidence that the next two basepairs are also broken and likely have a base flipped out of the helix in the pre and post-cleavage intermediate. It is predicted that an underwound pre-cleavage intermediate drives the reaction forward and ensures reaction completion.
Description
Keywords
TelA, Pre-Cleavage Intermediate, Telomere resolvase
DNA enzymes
agrobacterium tumefaciens
hairpin telomeres
DNA-protein interaction
Citation
Degree
Master of Science (M.Sc.)
Department
Medicine
Program
Microbiology and Immunology