Structure of the replication regulator Sap1 reveals functionally important interfaces

TY - JOUR

T1 - Structure of the replication regulator Sap1 reveals functionally important interfaces

AU - Jørgensen, Maria Louise Mønster

AU - Ekundayo, Babatunde

AU - Zaratiegui, Mikel

AU - Skriver, Karen

AU - Thon, Genevieve

AU - Schalch, Thomas

PY - 2018/12/1

Y1 - 2018/12/1

N2 - 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.

AB - 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.

U2 - 10.1038/s41598-018-29198-9

DO - 10.1038/s41598-018-29198-9

M3 - Journal article

C2 - 30026545

SN - 2045-2322

VL - 8

JO - Scientific Reports

JF - Scientific Reports

M1 - 10930

ER -