dc.contributor.advisor | Cortez, David | |
dc.creator | Rua Fernandez, Jorge | |
dc.date.accessioned | 2024-05-15T17:33:19Z | |
dc.date.available | 2024-05-15T17:33:19Z | |
dc.date.created | 2024-05 | |
dc.date.issued | 2024-03-21 | |
dc.date.submitted | May 2024 | |
dc.identifier.uri | http://hdl.handle.net/1803/18974 | |
dc.description.abstract | Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions in mammalian cells. HMCES (5-hydroxymethylcitosine, embryonic cell specific) is an evolutionarily conserved protein found at replication forks that covalently crosslink to AP sites in ssDNA, forming a DNA-protein crosslink (DPC). The HMCES-DPC shields ssDNA AP sites from endonucleases and TLS polymerase activity to help maintain genome integrity. However, how the HMCES-DPC is resolved in human cells and produces better outcomes for genome stability and organism fitness remains unknown. In my thesis project, I describe a system utilizing DNA polymerase alpha inhibitor CD437 to induce the accumulation of ssDNA and AP sites during DNA synthesis. Hence, I use CD437 as a tool to track and quantify the formation and resolution of HMCES-DPCs in human cells. Using this CD437 system, I show that the proteasome and SPRTN are not required to remove HMCES-DPC from human cells. However, mutation of conserved residue Glu127 at HMCES’s SRAP domain strongly delays HMCES-DPC removal in cells. Biochemically, I demonstrate that Glu127 catalysis reversal of the crosslink and how DNA structure conditions HMCES re-crosslink capacity. I also report that deficiency of this crosslink reversal affects cell viability in the absence or presence of DNA stress agents. Taken together, my data suggest that HMCES-DPC removal in human cells can be mediated by the auto-release of the crosslink, which is important to maintain cellular fitness. This mechanism provides a new strategy to protect temporally AP sites, possibly until DNA replication provides a better DNA substrate to repair AP sites in an error-free pathway. | |
dc.format.mimetype | application/pdf | |
dc.language.iso | en | |
dc.subject | HMCES, AP site, DPC, self-reversal, cellular fitness | |
dc.title | Understanding the mechanisms of HMCES DNA-protein crosslink removal and its importance in human cells | |
dc.type | Thesis | |
dc.date.updated | 2024-05-15T17:33:20Z | |
dc.type.material | text | |
thesis.degree.name | PhD | |
thesis.degree.level | Doctoral | |
thesis.degree.discipline | Biochemistry | |
thesis.degree.grantor | Vanderbilt University Graduate School | |
dc.creator.orcid | 0009-0009-3289-7469 | |
dc.contributor.committeeChair | Hiebert, Scott | |