Fig 2: Model of the role of DNA double strand break repair during SV40 DNA replication.(A) Tag translocates toward a nick in the DNA (orange dashed arrow), unwinding the helix. (B) Helicase activity moves past the nick and generates a one-sided DSB that recruits MRN and KU to the DSB termini. Wild-type conditions (blue arrow): (C) MRN binding recruits and activates ATM at the DSB. ATM phosphorylates CtIP to create a stable interaction with NBS1 [59] at the DSB. ATM-phosphorylated MRN-CtIP catalyzes the initial 5′ to 3′ end resection of the DSB resulting in loss of KU. Further phosphorylation of BLM, DNA2, and EXO1 by ATM facilitates mobilization of each protein to broken DNA and more robust end resection leading to the loss of KU at viral DNA. (D) The single-stranded DNA generated by end resection is bound by RPA. Replacement of RPA on resected 3′ tail with RAD51 facilitates HDR, and the DSB is repaired to yield two intact unit length viral genomes at the end of the replication cycle (E). ATM inhibited conditions (red arrow): (F) MRN binding recruits ATM to DSB termini. However, failure of ATM to phosphorylate and create binding sites for CtIP, BLM/DNA2, or EXO1 causes stable KU binding to blunt or short single-stranded DNA at the DSB termini. Bound KU recruits DNA-PKcs to the DSB. DNA-PKcs kinase activity is activated to commence NHEJ. The DSB is not repaired efficiently by NHEJ resulting in rolling circle replication and concatemer formation (G).

Fig 3: USP15 is phosphorylated during infection in a UL12-dependent manner. (A) 293T or (B–E) Vero cells were infected with KOS or AN-1 at an MOI of 10 and collected at 6 hours post-infection, unless indicated otherwise. (A and B) Cell lysates were separated by SDS-PAGE or with Phos-Tag acrylamide gels to visualize phosphorylated proteins and then immunoblotted with USP15, ICP4, and ICP8 antibodies. (C) Cell lysates were treated with lambda phosphatase (λPPase) for 30 minutes prior to separating proteins by SDS-PAGE and immunoblotting with USP15, Nbs1, and ICP8 antibodies. (D) Comparison of USP15 mobility by immunoblot during infection with KOS or AN-1. Where indicated, Vero cells were first transduced with lentivirus expressing UL12 prior to infection (+ UL12). (E) Infections were performed in the presence of a specific ATM inhibitor (ATMi) or viral helicase/primase inhibitor (H/Pi). All experiments were performed at least three times, and one representative immunoblot is shown in each panel.