RESEARCH IN PROGRESS SEMINAR 2018: Gavin Grant, PhD and Sourav Roy, PhD (UNC)

Gavin Grant, PhD
Postdoctoral Researcher
Department of Biophysics & Biochemistry
Jean Cook Lab

“Rushing through the morning- potential consequences of an abbreviated G1 phase.”

Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To precisely mark these transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create “PIP-FUCCI.”  With these tools, we made the unexpected observation that the apparent timing of APCCdh1 inactivation frequently varies relative to the onset of S phase. We demonstrate that APCCdh1 inactivation is not a strict pre-requisite for S phase entry. These precise markers for cell cycle phase boundaries uncover the sequences of molecular events at critical cell cycle transitions. 

Sourav Roy, PhD
Postdoctoral fellow
Department of Biochemistry & Biophysics
Charles Carter Lab

“Dissecting oligopeptide-oligonucleotide interactions: leads from laboratory evolution”

Interactions between oligonucleotides and proteins have important biological functions as seen in tRNA synthetases, polymerases, ribosomes, histones, etc. However, the origin of these complexes is obscure and demands extensive investigation. Carter and Kraut 1 showed from model building that the two polymers have stereochemically complementary structures, and suggested that complexes between RNA and peptide hairpins might have played a role in each polymer catalyzing biosynthesis of the other. We aim to use in-vitro selection to test this proposal and identify possible sequence dependences underlying the interaction between the β-hairpin peptides and RNA oligonucleotides. This is a challenging task, because it depends on binding between two potentially diverse libraries, which necessarily limits the concentration of any single species. To achieve high enough concentrations of individual species, we have devised several different sampling algorithms, of which the most successful appears to be the sample complex formation between a diverse RNA library and a sampled design of peptide hairpins (1015 for the RNA library and 11 peptides from a discrete set). After seven rounds of selection, we observed the enrichment of a sequence that exhibited association with a peptide having a net charge of –3. The peptide additionally has a cysteine residue at either end and the β-hairpin structure of the peptide in solution was confirmed using circular dichroism spectroscopy,which is further enhanced by the presence of DTT. In order to find the potential binding sites for the peptide in the RNA structure, we focused on a 16nt loop in the variable region of the sequence. Isothermal titration calorimetry suggests the binding of the peptide to the short loop with a dissociation constant of < 20 µM. In addition, we have also observed the binding of the peptide to the DNA sequence equivalent to the short RNA loop with a comparable affinity. We further designed several variants for the DNA loop sequence wherein the residues in either the stem/loop region or both are substituted. Preliminary studies have indicated that both stem and loop regions are necessary for binding. The thermodynamics of binding, obtained from ITC, imply that the interaction is held together by hydrophobic bonding. As these experiments need further validation, we are in the process of characterizing several other variants that are expected to establish the role of individual residues in the loop and the stem in peptide binding.

Reference:

1.       Carter, C.W., Jr.  and Kraut, J. (1974) Proc. Nat. Acad. Sci. USA, 71, 283-287.