Role of the RPA-Sgs1 interaction in stabilizing stalled replication forks

S phase is the period of the cell cycle when all genomic DNA is copied precisely twice. During this complex process, replication forks frequently encounter obstacles such as tightly bound protein-barriers or are challenged by genotoxic insults creating DNA damage. As a consequence replication forks stall and form fragile DNA structures that need to be stabilized and restarted in order to prevent DNA double strand break (DSB) formation and aberrant homologous recombination (HR). Therefore, the intra-S phase checkpoint, a sophisticated surveillance mechanism, is activated to restrain potential fork collapse and to regulate cell cycle progression, DNA repair and late origin firing. Two important proteins in stabilizing arrested replication forks are the checkpoint kinase Mec1 and the RecQ helicase Sgs1 in S. cerevisiae. It has been proposed that both pathways in maintaining fork integrity converge on replication protein A (RPA). In fact, RPA had been shown to recruit Mec1-Ddc2 to stalled replication forks and to bind Sgs1. Therefore, this PhD work aimed to study which impact the RPA-Sgs1 interaction has in stabilizing stalled replication forks in response to the replication fork inhibitor hydroxyurea (HU).
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.