Towards construction and validation of an ends-in recombination system in Escherichia coli

Homologous recombination is the primary DNA repair pathway in bacteria and it is immensely important in repairing DNA double strand breaks. Components of the homologous recombination pathway have been well conserved throughout evolution as an essential part of cell survival. Homologous recombination plays an important role in cellular processes like DNA repair as well as exchange of genetic information through chromosomal crossover. During homologous recombination, DNA strand exchange leads to formation of a heteroduplex joint between the invading and displaced DNA strands. This hetereoduplex joint is called a Holliday Junction. Resolution of the Holliday Junction proceeds via one of two pathways. In the presence of RuvC and/or RecG, Holliday Junction resolution proceeds via a “cut and paste” pathway where the invading DNA strand replaces a region of homologous DNA on the target DNA. In the absence of RuvC and RecG, Holliday Junction resolution takes place via a “copy and paste” pathway during which DNA synthesis needs to be primed at Holliday Junction intermediates formed during strand invasion. In an effort to separate this myriad of different requirements, I have attempted to develop a novel “ends-in” recombination assay system using E. coli as a model organism. This ends-in system would allow recombinant molecule formation by DNA synthesis of approximately 200 to 2000 bp size interval between the two converging ends of an invading linear dsDNA substrate oriented just like the greek letter Ù, but with the arms pointing inwards. In this study, a number of linear dsDNA assay templates were constructed and analyzed. All the constructs had two “arms” of homology to the chromosome pointing inwards i.e. in the ends-in orientation. Using this ends-in system, it was demonstrated that the presence of chi (Crossover Hotspot Initiator) sites was an important requirement for ends-in recombination in wild type E. coli cells. Our studies also showed that ends-in homologous recombination did not occur if chi sites were placed at or very near to the ends of the incoming linear dsDNA molecule, suggesting that the chi site recognition is efficient only if the incoming dsDNA has chi sites internal to the ends. Moreover, it was shown that neither RuvC nor RecG were required for successful recombinant product formation using the ends-in assay. This finding reinforces previous observations that suggest the idea that Holliday Junctions can be resolved independent of both RuvC and RecG

Coupling Protein Catabolism to Lifespan and Reproduction in Caenorhabditis elegans

There is an undisputable link between aging and reproduction and it has both puzzled and fascinated biologists for decades. Evolutionary biologists suggest that the rate of aging depends on the complex tug-of-war between maintenance of the soma and maintenance of the germ cells. The former is essential for longevity while the latter is essential for transmitting genetic information from parents to progeny. Any perturbation in the fertility and fecundity of C. elegans influences lifespan and vice-versa. Interestingly, germline deficient animals have increased resistance to environmental and proteotoxic stress. All eukaryotic cells reproduce for a finite amount of time before irreversibly ceasing reproduction, a phenomenon called reproductive senescence. Recent research has been aimed at trying to elucidate the genetic factors that regulate reproductive and post-reproductive lifespan. As part of this ongoing process, the initial part of my work aims to characterize the increased reproductive lifespan of a C. elegans mutant that is deficient for the gene rer-1. I further demonstrate that rer-1 mutants show a higher level of autophagy which is responsible for the enhanced reproductive lifespan of these mutants. Aging is thought to be a stochastic process, and cessation of reproduction is one of the biological hallmarks of aging. Although both reproduction and aging are well studied processes, there is very little mechanistic understanding of how these processes are connected and coordinated. The latter part of this study aims to answer some of these questions about the cross-talk between the germ cells and somatic tissue in C. elegans using a panel of sterile hermaphrodites impaired for specific stages of reproduction. My findings show that cessation of fertilization triggers a signalling cascade from the germ cells to the soma and that this signalling is brought about by steroid hormones, presumably synthesized by the somatic gonad. In actively reproducing worms, the forkhead transcription factor DAF-16 drives expression of vha genes which encode a multi-subunit proton pump that is responsible for maintaining lysosomal acidity. Nuclear exclusion of DAF-16 in post-reproductive worms co-ordinately reduces the expression of vha genes resulting in lysosomal alkalinization that culminates in acute loss of fitness and contributes to organismal senescence. My data shows a plausible mechanistic pathway by which lysosomal acidity is regulated via gonad to soma signalling in young (reproducing) animals