Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Microbiology

Year Degree Awarded

2019

Month Degree Awarded

February

First Advisor

Michele M Klingbeil

Second Advisor

Yasu S. Morita

Third Advisor

M. Sloan Siegrist

Fourth Advisor

Arthur Günzl

Subject Categories

Molecular Biology | Parasitology | Pathogenic Microbiology

Abstract

DNA is the substrate of many cellular processes including DNA replication, transcription and chromatin remodeling. These processes are coordinated to maintain genome integrity and ensure accurate duplication of genetic and epigenetic information. Genome-wide studies have provided evidence of the relationship between transcription and DNA replication timing. A global analysis of DNA replication initiation in T. brucei showed that TbORC1 (subunit of the origin recognition complex, ORC) binding sites are located at the boundaries of transcription units. Although recent studies in T. brucei indicate functional links among DNA replication and transcription, the underlying mechanisms remain unknown. In this study, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system.The iPOND approach relies on labeling newly replicated DNA with the thymidine analog EdU (5-ethynyl-2′-deoxyuridine), which contains an alkyne functional group that enables the cycloaddition of a biotin azide. This click chemistry reaction yields a stable covalent linkage, facilitating streptavidin capture of cross-linked biotinylated DNA-protein complexes. First, we described how we adapted the iPOND protocol in T. brucei cells to generate a suitable sample for mass spectrometry analysis (Chapter 2). We performed MS label-free quantification to determine which proteins are enriched in an active replication fork in T. brucei (Chapter 3). We identified 410 proteins, including key DNA replication factors and proteins associated with transcription, chromatin organization, DNA repair and mRNA splicing. Around 25% of the proteins identified were of unknown function that might have the potential to be essential trypanosome-specific replication proteins. Additionally, we characterized two proteins from our iPOND-derived protein list (Chapter 3). These are a protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of unknown function (Tb927.3.5370). Both revealed nuclear localization. Tb927.10.7990 proved to be essential since its silencing caused a growth defect and impaired DNA replication. However,Tb927.3.5370 appeared to be a dispensable gene. We propose nucleosomes are assembled close to the replication fork followed by RNA pol II recruitment, transcription, and co-transcriptional RNA splicing. Further studies are needed to determine how these processes are linked and co-regulated, and how rapidly they are initiated during DNA replication.

Sup Table 1.xlsx (259 kB)

Share

COinS