Bacteriophage T4 Initiates Bidirectional DNA Replication through a Two-Step Process  Karyn Goudie Belanger, Kenneth N Kreuzer  Molecular Cell  Volume 2, Issue 5, Pages 693-701 (November 1998) DOI: 10.1016/S1097-2765(00)80167-7

Figure 1 Schematic of the uvsY Region of the T4 Genome (A) shows the position of the middle mode promoter (PM), the minimal origin, the uvsY coding sequence, two downstream ORFs, and the transcriptional terminator. (B) shows the position of restriction sites surrounding ori(uvsY) that are important in this study. The two SwaI sites in bold (*) were engineered into the chromosome and are only present in particular mutant phage strains. The SwaA site could only be cleaved by the SwaI isoschizomer, SmiI. Molecular Cell 1998 2, 693-701DOI: (10.1016/S1097-2765(00)80167-7)

Figure 2 2D Gel Analysis of the ori(uvsY) Region, a Time Course Genomic replication assays were conducted with phage strain K10 as described in the Experimental Procedures. DNA samples were prepared at 7 min (A), 9 min (B), 11 min (C), and 13 min (D) postinfection. The first dimension of each 2D gel is represented horizontally (left to right) and the second dimension vertically (top to bottom). The arrows point to the comet region on the simple Y arc. The vertical streak emanating from the monomer spot in (C) and (D) was often observed at these time points in an infection but is uncharacterized (we suspect it is a gel artifact). All DNA samples were cleaved with both PacI (cuts at 113.762 kb) and SwaI (cuts at 118.342 kb) prior to electrophoresis. Each panel is a Southern blot of the 2D gel, using an ori(uvsY) PacI-SwaI fragment probe (113.762–118.342 kb). Molecular Cell 1998 2, 693-701DOI: (10.1016/S1097-2765(00)80167-7)

Figure 3 2D Gel Analysis of the ori(uvsY) Region Genomic replication assays were conducted with phage strains K10 (A), K10-608 (B), K10-116 (C), K10-KB1 (D), K10-KB2 (E), K10-Term (F), and K10-GC (G) phage as described in Figure 2. The schematic map above each 2D gel indicates which mutation (if any) is present in the phage. The arrows point to the comet region on the simple Y arc. Molecular Cell 1998 2, 693-701DOI: (10.1016/S1097-2765(00)80167-7)

Figure 4 2D Gel Analysis of Mutant Infections Genomic replication assays and 2D gel analysis were conducted with phage strains K10 (A), K10-43am (B), K10-uvsX (C), K10-46am (D), K10 (E), and K10-rnhΔ (F) as described in Figure 2. For (E) and (F), the DNA samples were harvested at 9 min postinfection. The E. coli host used for the infection in (F) was MIC1044 (deficient in RNase H1 and 5′ to 3′ exonuclease of DNA Pol I). Molecular Cell 1998 2, 693-701DOI: (10.1016/S1097-2765(00)80167-7)

Figure 5 The Comet Maps to the Origin Transcript Genomic replication assays were conducted with phage strains K10-SwaA (A), K10-SwaA (B), K10-SwaAB (C), and K10-SwaB (D) (see Figure 1A). The sample in (A) was digested with PacI and SwaI, while the samples in (B), (C), and (D) were digested with SwaI and SmiI prior to electrophoresis (SmiI is an isoschizomer of SwaI that cleaves the SwaA site effectively). (A) and (B) were probed with the ori(uvsY) PacI-SwaI fragment (113.762–118.342 kb) while (C) and (D) were probed with the ori(uvsY) XbaI-EcoRI fragment (113.057–116.338 kb). Molecular Cell 1998 2, 693-701DOI: (10.1016/S1097-2765(00)80167-7)

Figure 6 Northern Analysis of Origin Transcript RNA was prepared from AB1 cells infected with T4 strain K10 (lane 1), K10-GC (lane 2), and K10-608 (lane 3). The infected cells were harvested at 7 min postinfection. The RNA was run on a formaldehyde gel, blotted to nylon, and probed with an oligonucleotide that hybridizes to the uvsY transcript (A). The blot was then stripped and reprobed with an oligonucleotide that hybridizes to the gene 32 transcript (B). Molecular Cell 1998 2, 693-701DOI: (10.1016/S1097-2765(00)80167-7)

Figure 7 Model for Origin Initiation in Phage T4 (A) depicts the proposed pathway for initiating rightward replication, and (B) depicts assembly of the leftward fork. See text for details. Molecular Cell 1998 2, 693-701DOI: (10.1016/S1097-2765(00)80167-7)