Premium
Characterizing the Thermodynamic Stability of the Holliday Junction Through Spectroscopic Techniques
Author(s) -
Taylor Nicholas Brown,
Mukerji Ishita,
Savage Rachel,
McDonald Julie Lynn,
Qiu Rong
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06912
Subject(s) - holliday junction , base pair , duplex (building) , chemical stability , dna , biophysics , crystallography , chemistry , chemical physics , materials science , homologous recombination , biology , physics , thermodynamics , genetics
The Holliday junction is a four‐way DNA structure formed during the process of double strand break repair and DNA recombination in meiosis. Branch migration of the junction is a vital molecular process for the transfer of genetic information from parents to offspring. Understanding this process relative to base pair composition can provide a better picture of the overall structure of the junction and its connection to short inverted repeats that have been linked to chromosome instability. We used a fluorescent nucleoside analog, 4‐amino‐6methyl‐8‐(2‐deoxy‐beta‐d‐ribofuranosyl)‐7(8H)‐pteridone (6‐MAP), to obtain site‐specific information about the melting process, through incorporation of the probe at distinct sites throughout a 34 bp immobile junction. Under our conditions, the junction primarily adopts a conformation where the arms stack co‐axially on each other. We have examined the stability of the junction arms by incorporating the probe at equivalent positions on each arm. From these measurements we have found that the junction follows a 'pseudo‐duplex' model of denaturation, in which one of the coaxially stacked pair of arms melts at a lower temperature relative to the other. Our results also point to a sequential melting pattern in which positions at the end of the arms and the center destabilize before the interior of the arms. The relative proximity to the junction center appears to be one of the factors affecting stability. Our current research investigates how sequence homology influences the stability of the junction, specifically whether the thermodynamic characteristics of an immobile junction are predictive for a mobile one. We have developed a mobile junction with 10‐bp homology in the central region. Previous research by (Voth A.R., Hays F.A., Ho P.S. (2007) Directing macromolecular conformation through halogen bonds. Proc. Natl. Acad. Sci. USA.; 104 :6188–6193. doi: 10.1073/pnas.0610531104) has suggested that macromolecular conformations can be directed through the use of halogen bonds. Specific incorporation of brominated uracils in the sequence of two DNA strands of a mobile Holliday junction was shown to bias the structure to a single conformation in crystallographic experiments. Combined spectroscopic data from both the immobile and mobile junction will inform coarse‐grained modeling of the Holliday junction. Our experimental data combined with computational modeling furthers our current understanding of this DNA structure as it relates to branch migration and exchange of genetic information.