ARCHAEAL DNA REPLICATION PROTEINS: MEMBERS AND FUNCTIONS
| dc.contributor.advisor | Kelman, Zvi | en_US |
| dc.contributor.author | Li, Zhuo | en_US |
| dc.contributor.department | Cell Biology & Molecular Genetics | en_US |
| dc.contributor.publisher | Digital Repository at the University of Maryland | en_US |
| dc.contributor.publisher | University of Maryland (College Park, Md.) | en_US |
| dc.date.accessioned | 2014-02-08T06:31:17Z | |
| dc.date.available | 2014-02-08T06:31:17Z | |
| dc.date.issued | 2013 | en_US |
| dc.description.abstract | The mechanism of DNA replication in archaea, the third domain of life, has been studied for more than two decades using biochemical, structural and bioinformatic approaches. Historically, many of the proteins that participate in archaeal replication were identified via similarity to enzymes needed for DNA replication in bacteria and eukarya. This study uses a different approach to identify new factors that may be involved in replication. Genetic tools developed for the thermophilic archaeon Thermococcus kodakarensis were used to identify new replication factors that could not be recognized through in silico methods. First, a network of proteins that may participate in replication was identified using in vivo tagging of known replication enzymes. Following affinity purification the proteins that co-purified with the tagged enzymes were identified using mass spectrometry. This study describes the identification of a number of new putative replication factors. Next, the biochemical properties of two proteins identified in the screen were characterized. One, the product of gene TK1525, was identified via its interaction with the GINS complex. This protein was predicted to be an archaeal homologue of the bacterial RecJ nuclease. It was found that the protein is a processive, manganese-dependent, single strand DNA-specific exonuclease. The protein was designated GAN for GINS-associated nuclease. GAN forms a complex with GINS and also interacts with the archaeal-specific DNA polymerase D in vivo. Subsequent bioinformatic analysis suggested that GAN may be the archaeal homologue of the eukaryotic Cdc45 protein. The second protein characterized is the product of TK0808. This protein was identified via its interactions with proliferating cell nuclear antigen (PCNA). The protein, upon binding to PCNA, inhibits PCNA-dependent activities. The protein was therefore designated TIP for Thermococcales inhibitor of PCNA. While most proteins that interact with PCNA do so via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA, TIP neither contains the canonical PIP motif nor interacts with PCNA via the IDCL. These findings suggest a new mechanism for PCNA binding and suggest a new mechanism to regulate PCNA-dependent activities. | en_US |
| dc.identifier.uri | http://hdl.handle.net/1903/14888 | |
| dc.language.iso | en | en_US |
| dc.subject.pqcontrolled | Biology | en_US |
| dc.subject.pqcontrolled | Microbiology | en_US |
| dc.subject.pqcontrolled | Molecular biology | en_US |
| dc.subject.pquncontrolled | Archaea | en_US |
| dc.subject.pquncontrolled | DNA replication | en_US |
| dc.title | ARCHAEAL DNA REPLICATION PROTEINS: MEMBERS AND FUNCTIONS | en_US |
| dc.type | Dissertation | en_US |
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