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5-hydroxymethylcytosine and endonuclease G as regulators of homologous recombination

dc.contributor.authorVander Zanden, Crystal M., author
dc.contributor.authorHo, P. Shing, advisor
dc.contributor.authorPeersen, Olve, committee member
dc.contributor.authorDi Pietro, Santiago, committee member
dc.contributor.authorFisk, Nick, committee member
dc.date.accessioned2017-09-14T16:04:42Z
dc.date.available2018-09-12T16:04:38Z
dc.date.issued2017
dc.description.abstractHomologous recombination (HR) is a necessary biological process for all living organisms, and it is especially important for repairing damaged DNA. Improper HR results in DNA damage-related diseases, notably increased likelihood of cancer when HR regulators, such as the human BRCA1 gene, are impaired. HR is also a tool for biotechnology, giving scientists the power to easily delete or mutate genes and study the effects of those modifications. Recently, the epigenetically modified nucleotide 5-hydroxymethylcytosine (5hmC) was found to regulate vertebrate HR via interaction with the protein endonuclease G (EndoG). In this dissertation, I use biochemical/biophysical methods to elucidate the interaction between 5hmC and EndoG, thus working towards understanding their roles as regulators of recombination. I find that 5hmC forms a unique hydrogen bond to stabilize Holliday junctions, the four-stranded DNA intermediate in HR. 5hmC also induces a global structure change to the junction, increasing protein access to the junction crossover and providing potential for either direct or indirect readout of 5hmC. Further connecting EndoG with recombination, we present the first evidence that EndoG preferentially binds and cleaves Holliday junction DNA, implicating a role for EndoG as a resolvase. I demonstrate that EndoG recognizes 5hmC in the junction context and observe unique cleavage products from EndoG interaction with 5hmC-junctions. These results suggest that EndoG may have a previously unrecognized junction resolvase function and, in this way, play a more direct role in recombination than simply creating double-stranded breaks in duplex DNA to initiate the HR mechanism. Finally, I present a unique structural feature of vertebrate EndoG that we hypothesize is the basis for 5hmC recognition. I present the structure of mouse EndoG and propose that a two amino acid deletion, conserved in vertebrate EndoG sequences, is associated with unraveling of an α-helix. This structural perturbation positions amino acid side chains to confer 5hmC-sensing ability to all vertebrate EndoG. I expect that these deletion mutations and resulting structural effects co-evolved with the appearance of 5hmC in vertebrate genomes to give EndoG an additional function of recognizing 5hmC in the cell. Overall this work is building onto the understanding of 5hmC and EndoG as markers and regulators of recombination.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierVanderZanden_colostate_0053A_14284.pdf
dc.identifier.urihttps://hdl.handle.net/10217/183918
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectcrystallography
dc.subjectrecombination
dc.subjectendonuclease G
dc.subject5-hydroxymethylcytosine
dc.title5-hydroxymethylcytosine and endonuclease G as regulators of homologous recombination
dc.typeText
dcterms.embargo.expires2018-09-12
dcterms.embargo.terms2018-09-12
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineBiochemistry and Molecular Biology
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)

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