Author ORCID Identifier
https://orcid.org/0000-0002-0873-8937
Date of Award
27-1-2025
Document Type
Thesis
School
School of Chemical & Biotechnology
First Advisor
Dr. Anjana Badrinarayanan
Second Advisor
Dr.R.B.Chandra Mohan
Keywords
Homology Search, Recombination, RecA, SMC Proteins, DNA Repair
Abstract
The most deleterious form of DNA damage is the formation of a double-strand break (DSB). If left unrepaired or repaired incorrectly, DSBs can result in genome rearrangements, mutations, or even cell death. Hence, cells across domains of life have resorted to homologous recombination (HR) for the faithful repair of DSBs. A critical step in homologous recombination is the search for the intact homologous sequence by the break ends, termed ‘homology search’. This process is not well understood in vivo, especially when the break site and intact homologous template are not positioned adjacently.
Spatial reorganization of chromosomes is maintained by structural maintenance of chromosome (SMC) proteins, some of which also play a role in DNA repair. This makes SMCs potential candidate proteins involved in homology search. Hence, we investigated the bacterial SMC protein, RecN, which is essential for DNA repair but its mechanism of action remains unexplored in vivo. To study ‘homology search’ involving RecN in living cells, we used Caulobacter crescentus as a model organism.
Caulobacter served as a promising model system due to a previously established protocol to study the repair of a single DSB. In this system, the spatial organization of the Caulobacter chromosome positions the break site and the intact homologous sequence ~1-2μm apart, allowing us to study long-distance homology search. We induced a single DSB using the I-SceI endonuclease and observed the dynamics of break ends coated with RecA nucleoprotein filaments during homology search and repair. To specifically study homology search and homology search followed by repair, we used non-replicating swarmer (1N) and pre-divisional (2N) cells, respectively.
Our first key observation was the directional translocation of RecA nucleoprotein filaments along the cell length. They performed multiple pole-to-pole traversals before finding the intact homologous sequence. These traversals were independent of the presence of the intact homologous chromosome harboring the template for repair. Most interestingly, these translocations were abolished in the absence of recN or its ATPase activity.
During traversals, RecA nucleoprotein filaments also show variations in morphology, especially length. We observed that this fluctuation in filament length in wild-type cells is significantly reduced in the absence of recN. Additionally, in the ATP hydrolysis mutant of RecN, the RecA filament lengths were noticeably shorter compared to those observed in the absence of recN or in wild-type cells, suggesting that ATPase activity might be essential for the spatial orientation of RecA nucleoprotein filaments during homology search.
Finally, we observed that the rate of translocation is contingent on the chromosomal content. In swarmer cells (1N), RecA nucleoprotein filaments complete pole-to-pole traversals in less than half the time compared to the traversal time observed in predivisional cells (2N). Taken together, we establish that SMC proteins are essential for the translocation of RecA nucleoprotein filaments to carry out homology search and repair during DSB. This work transitions into the next chapter of understanding homology search, where we will aim at unravelling the role of RecN in maintaining directionality during the translocation of RecA nucleoprotein filament.
Recommended Citation
Chimthanawala, Afroze Abdehaque Mr, "Role of an SMC-like protein, RecN, in RecA-mediated homology search during double-strand break repair via homologous recombination" (2025). Theses and Dissertations. 14.
https://knowledgeconnect.sastra.edu/theses/14