Figure 1.
SV40 chromatin replication results in DNA damage signaling.
(A) Representative images of chromatin-bound Tag and host DNA replication proteins in SV40- and mock-infected (inset) BSC40 cells at 48 hpi. (B) Features of the SV40 genome and the insertion site of pMini vector [34]. Mutation of Tag residue 474 from D to N abrogates helicase activity [34]. The defective SV40 origin mutant, In-1, features an insertion of a single GC bp in the center of the viral origin allowing Tag binding, but not origin activation [35]. (C, D, E) BSC40 cells transfected with the indicated pMini SV40 plasmids were analyzed by (C) western blot after 24 h, (D) Southern blot of low molecular weight DNA after 48 h [34], [73], or (E) immunofluorescence microscopy of chromatin-bound proteins. In (D), SV40 or Mitochondrial probe signal is denoted by SV40 or Mito, respectively. Scale bars in (A) and (E), 10 µm.
Figure 2.
ATM inhibition during viral DNA replication disrupts viral replication centers.
(A) Experimental scheme for treatment of cells with inhibitor during phases of a 48 h SV40 infection. Early: inhibitor present from −0.5 to 20 hpi. Late: inhibitor present from 20 to 48 hpi. DMSO and Full: solvent or inhibitor, respectively, present from −0.5 to 48 hpi. (B) Western blot of cells treated with Ku-55933 as described in (A). (C) Immunofluorescence of cells treated with Ku-55933 as described in (A) and fixed at 48 hpi. Scale bars, 10 µm. (D) Tag staining patterns, as in (C), were quantified. Graph shows the average of 3 independent experiments.
Figure 3.
Ku-55933 treatment during viral DNA replication increases aberrant DNA structures.
(A) Southern blot of DNA from SV40 infected BSC40 cells in the presence of DMSO or Ku-55933. DMSO or Ku-55933 was present from 30 min prior to infection until cell collection timepoint. M represents Mock-infected cells. (B) Each normalized monomer SV40 form I, II, and III product in (A) was graphed as a fraction of the corresponding normalized monomer produced at 72 hpi in the DMSO control infection. (C) Graph of the percentage of DNA products represented by concatemers in panel (A). (D) Southern blot of SV40 DNA replicated in the presence of Ku-55933 during phases of a 48 h infection in BSC40 cells as explained in Figure 2A. (E) Quantification of total and monomeric SV40 DNA signal normalized to DMSO control from southern blots as in (D). (F, G) Graph of DNA structures (monomer: F and DNA Structure: G) accumulating on southern blots as in (D). Graphs in (E–G) represent 3 to 4 independent experiments.
Figure 4.
ATM inhibition increases recombination and unidirectional replication.
(A) Diagram of neutral 2 d gel electrophoresis arcs generated from digested SV40 DNA. (B) Replicating viral DNA extracted from unperturbed SV40-infected cells consists primarily of circular, late replication intermediates called late Cairns intermediates. Digestion of late Cairns intermediates with BglI yields large double Ys, whereas BamHI digestion yields large bubbles. (C, D, E, F) Southern blot of neutral 2 d gel of BglI-cleaved DNA replicated in the presence of DMSO (C, D) or Ku-55933 (E, F) during the late phase of a 48 h SV40 infection in BSC40 cells. DNA was cleaved within the viral origin of replication with BglI (C, E) or the region of fork convergence with BamHI (D, F). The red star denotes an arc representing strand invasion events (D-loops) or highly branched molecules [38], [39]. On the simple Y arc in (F), S denotes a replication stall point near the viral origin of replication. Dashed boxes denote regions of each arc quantified in (H) and (I). (G) Concatemers of SV40 DNA that accumulated when ATM was inhibited can arise by either replication- (top) or recombination- (bottom) dependent rolling circle replication. Digestion of replication-dependent rolling circles with BglI or BamHI results in simple Ys of all sizes. Digestion of recombination-dependent rolling circles creates D-loops of all sizes. (H) Graph of DNA signal present on simple Y, double Y, X structure, or D-loop arc divided by DNA signal in the double Y arc from DNA digested with BglI. (I) Graph of DNA signal from BamHI digested DNA in simple Y, bubble, X structure, or D-loop arc divided by DNA signaling in the bubble arc. Each graph in (H) and (I) represents the average of 3 to 4 independent experiments.
Figure 5.
ATR is crucial for SV40 chromatin replication.
(A) Scheme for application of ATRi during phases of a 48 h SV40 infection. (B) Southern blot of DNA replicated in BSC40 cells when ATRi was present during phases of a 48 h SV40 infection described in (A). (C) Graph of total viral or SV40 monomer DNA signals normalized to SV40 DNA replicated in the presence of DMSO from southern blots as shown in (B). (D, E) Graph of of monomer (D) or aberrant (E) structure(s) accumulated as a result of ATR inhibition from southern blots as shown in (C). Each bar in (C–E) shows the average from 3 to 4 independent experiments.
Figure 6.
ATR inhibition results in fork stalling and breakage of converging forks.
(A) Schematic of replication intermediate migration patter on a neutral 2 d gel generated from digested SV40 DNA. (B, C, D, E) Southern blot of neutral 2 d gel electrophoresis of BglI- (B, C) or BamHI-cut (D, E) DNA from SV40-infected BSC40 cells exposed to DMSO (B, D) or ATRi (C, E) during the late phase of SV40 infection as described in Figure 5A. (F) Diagrams of replication intermediates on a simple Y arc produced when ATR was inhibited. BamHI (green) and BglI (orange) sites are denoted by colored lines. I. Replication initiates at the origin and proceeds bidirectionally producing theta replication intermediates. II. Replisomes continue replication until one encounters a replication block (red triangle) causing one stalled fork. III. The stalled replication fork is closest to orange BglI site (viral origin of replication). The functional replisome continues replication and converges with the stalled replication fork. IV. One-sided DSB forms at the replicating fork of late Cairns intermediate shown in (III) as it translocates toward the stall site. V. Simple Y created by digestion of the broken late Cairns intermediate shown in (IV) with BglI or BamHI. VI. Diagram of the predicted outcome of the simple Y shown in panel (V) following neutral 2 d gel electrophoresis and southern blotting. The stall point on the simple Y arc (light green circle) corresponds to the simple Y in panel (V).
Figure 7.
Model of ATM and ATR functions in SV40 DNA replication.
(I) Tag initiates viral DNA replication at the viral origin of replication (blue) and the two replication forks progress bidirectionally (red arrowheads). For simplicity, proteins are not shown. (II) Viral DNA replicates quickly until the forks converge to form a late Cairns intermediate (III), which slowly completes replication. (IV) Topoisomerase IIα decatenates fully replicated DNA molecules, yielding two form I daughter molecules. (V) When ATM is inhibited, a one-ended double strand break at a replication fork leads to loss of the replication machinery, while the other fork continues to replicate DNA, generating a rolling circle (VI). (VII) ATM kinase activity facilitates the repair of one-ended double strand breaks. (VIII) When ATR is inhibited, a stalled replication fork remains stable until a functional replication fork approaches it, generating a broken replication intermediate (IX). (X) ATR kinase activity facilitates convergence of moving fork with the stalled fork. We suggest that in the presence of ATM and ATR, repair proteins act on the defective intermediates V and IX to reassemble an intermediate with two functional forks.