Hegnauer, Anna Maria. Role of the RPA-Sgs1 interaction in stabilizing stalled replication forks. 2011, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9704
During the first part of this PhD project, I have determined the interaction site between Sgs1 and the single strand binding heterotrimer RPA. On Sgs1, I have identified an unstructured, acidic region N-terminal to the helicase domain, which binds Rpa70 and had not been characterized before. I have created a new mutant, sgs1-r1, which completely disrupts Rpa70 interaction by two hybrid analysis. Indeed, we found that sgs1-r1 partially displaces DNA pol α from HU-stalled replication forks. However, in contrast to sgs1Δ, sgs1-r1 behaves epistatic to the S-phase specific mec1-100 mutant in response to HU, indicating that both factors act on the same pathway for replisome stability. Our data suggests that RPA-binding and helicase function of Sgs1 are necessary for full DNA pol α association at HU-arrested replication forks. Furthermore, we demonstrate that the same Sgs1 region that interacts with RPA is also a Mec1 target in vitro and is important for Rad53 activation after exposure to HU.
The main binding site on RPA was mapped to the N-terminal oligonucleotide binding (OB) fold of the largest RPA subunit, Rpa70. To gain structural insights, we have solved the structure of the N-OB fold of S. cerevisiae Rpa70 (this was performed by M. Vogel and P. Amsler in collaboration with N. Thomae’s laboratory). Despite low sequence conservation, the crystal structure of yeast Rpa70(3-133) displays high 3D conservation with the N-OB fold of human RPA70. It also consists of a five-stranded ß-barrel, capped by short α-helices and a basic cleft in the center. This cleft has been reported to mediate different protein-interactions in human cells. Therefore, we made use of the rfa1-t11 mutant, which carries a charge reversal mutation pointing towards this basic cleft. Indeed, rfa1-t11 partially disrupts Sgs1 binding as monitored by two-hybrid analysis. In addition, rfa1-t11 affects DNA pol α association at HU-stalled replication forks and displays a genome-wide replication defect in response to replication stress. These phenotypes for rfa1-t11 are stronger than for sgs1Δ, which indicates that only a fraction can be assigned to the loss of Sgs1 binding. However, we observe an epistatic relationship between rfa1-t11 and proteins involved in homologous recombination (HR) such as mre11 and rad51. We therefore suspect that impaired HR in rfa1-t11 cells might be the reason for the failure to restart DNA synthesis at stalled or collapsed replication forks.
|Committee Members:||Schär, Primo-Leo|
|Faculties and Departments:||09 Associated Institutions > Friedrich Miescher Institut FMI|
|Bibsysno:||Link to catalogue|
|Number of Pages:||157 S.|
|Last Modified:||30 Jun 2016 10:42|
|Deposited On:||15 Dec 2011 09:24|
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