Abstract
The mechanism by which specific protein-DNA complexes induce programmed replication fork stalling in the eukaryotic genome remains poorly understood. In order to shed light on this process we carried out structural investigations on the essential fission yeast protein Sap1. Sap1 was identified as a protein involved in mating-type switching in Schizosaccharomyces pombe, and has been shown to be involved in programmed replication fork stalling. Interestingly, Sap1 assumes two different DNA binding modes. At the mating-type locus dimers of Sap1 bind the SAS1 sequence in a head-to-head arrangement, while they bind to replication fork blocking sites at rDNA and Tf2 transposons in a head-to-tail mode. In this study, we have solved the crystal structure of the Sap1 DNA binding domain and we observe that Sap1 molecules interact in the crystal using a head-to-tail arrangement that is compatible with DNA binding. We find that Sap1 mutations which alleviate replication-fork blockage at Tf2 transposons in CENP-B mutants map to the head-to-tail interface. Furthermore, several other mutations introduced in this interface are found to be lethal. Our data suggests that essential functions of Sap1 depend on its head-to-tail oligomerization.
Highlights
As replication forks progress along the genome, they face many challenges including DNA damage lesions, strand breaks and DNA binding proteins which may cause the replication fork to pause or stall
As replication forks encounter the Tus-Ter complex they displace Tus from Ter sites when approaching from the permissive face of the complex, in the opposite orientation replication forks encounter the non-permissive face of the complex and are halted until replication terminates from the other side
Fork blockage is mediated by a ‘molecular mouse trap mechanism’ in which a cytosine in the Ter site flips into a specific pocket on Tus during strand separation as the oncoming helicases approach the non-permissive face of the complex[5]
Summary
As replication forks progress along the genome, they face many challenges including DNA damage lesions, strand breaks and DNA binding proteins which may cause the replication fork to pause or stall. Sequence comparison between Ter[1] and SAS1 reveals that the Sap[1] binding motifs occur as inverted repeats at SAS1 and as direct tandem repeats at Ter[1] (Fig. 1b)[11] This implies that a Sap[1] dimer is able to interact with its binding sites in two modes in which the same face of the complex is exposed to the oncoming replication fork when bound to SAS1 and two different faces of the protein are exposed when bound to Ter[1], suggesting different intramolecular interactions between Sap[1] monomers when bound to Ter[1] or SAS1. We present the crystal structure of the Sap[1] DNA binding domain which allows us to model Sap[1] bound to DNA in its proposed replication fork stalling orientation based on structural homology searches and biochemical data
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