SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase ¿-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that while Tag(131-627) (V350E/P417D) and Tag(131-627) (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag(1-708) and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation. This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, grant TE-671/4-2 to IT) and from the Else Kröner-Fresenius-Stiftung (EKFS 2013_A215) and the PML Consortium (Washington, USA; RIB1099) to HPN. The authors thank Patricia Nyland for expert technical assistance and Dr Lars Schönemann (Recombinant Protein Expression Facility, University of Würzburg) for his expert purification of wild type and mutant Tag131-627. We acknowledge Angela Borst for her contributions to AFM data collection and analyses, and Maciej Doczyk (NUI Galway) for his graphical work. We would also like to acknowledge the FP7 WeNMR (project# 261572), H2020 West-Life (project# 675858), the EOSC-hub (project #777536) and the EGI-ACE (project# 101017567) European e-Infrastructure projects for the use of their web portals, which make use of the EGI infrastructure with the dedicated support of CESNET-MCC, INFN-PADOVA-STACK, INFN-LNL-2, NCG-INGRID-PT, TW-NCHC, CESGA, IFCA-LCG2, UA-BITP, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, Taiwan and the US Open Science Grid. peer-reviewed