Multiple origins of replication contribute to a discontinuous pattern of DNA synthesis across the T4 genome during infection

Abstract

Chromosomes provide a template for a number of DNA transactions, including replication and transcription, but the dynamic interplay between these activities is poorly understood at the genomic level. The bacteriophage T4 has long served as a model for the study of DNA replication, transcription, and recombination, and should be an excellent model organism in which to integrate in vitro biochemistry into a chromosomal context. As a first step in characterizing the dynamics of chromosomal transactions during T4 infection, we have employed a unique set of macro array strategies to identify the origins of viral DNA synthesis and monitor the actual accumulation of nascent DNA across the genome in real time. We show that T4 DNA synthesis originates from at least five discrete loci within a single population of infected cells, near oriA, oriC, oriE, oriF, and oriG, the first direct evidence of multiple, active origins within a single population of infected cells. Although early T4 DNA replication is initiated at defined origins, continued synthesis requires viral recombination. The relationship between these two modes of replication during infection has not been well understood, but we observe that the switch between origin and recombination-mediated replication is dependent on the number of infecting viruses. Finally, we demonstrate that the nascent DNAs produced from origin loci are regulated spatially and temporally, leading to the accumulation of multiple, short DNAs near the origins, which are presumably used to prime subsequent recombination-mediated replication. These results provide the foundation for the future characterization of the molecular dynamics that contribute to T4 genome function and evolution and may provide insights into the replication of other multi origin chromosomes.

Figure 1

The T4 genomic macroarray. Although the terminally redundant, 172 kb T4 chromosome is linear, the ends are is circularly permutated, and there are no fixed telomeres. This results in a circular genetic map as depicted here. The location and position of PCR fragments included in the genomic array are indicated with yellow tabs with identifying numbers that correspond to the PCR loci (LP numbers) listed in Table 1. Putative T4 origins of DNA replication have been placed on the map as reported previously , and are indicated with green lollipops and identifiers (a, b, ect.). Major open reading frames (>100 amino acids) are indicated with arrows. These were placed on the genetic map using pDRAW32 and were color coded to indicate the timing of transcription, green, early; yellow, middle; and red, late transcripts based on Luke et al . Additionally, the position of two smaller T4 late genes soc and rI.-1 are indicated with red arrows near oriA and oriC, respectively.

Figure 2

T4 DNA synthesis during wt and recombination deficient infections. The increase in T4 genomes over the course of infection was monitored by hybridization to viral T4 DNA as described in Materials and Methods. (A) E. coli BL21(DE3) cells were infected at a multiplicity of five viruses per cell with either wt T4D (n=6), uvsX mutant (recA homologue, n=2), or gene59 mutant (helicase loader, n=5), where n is the number of independent experiments. (B) The first 15 minutes of the infection in (A) are plotted on an expanded scale. (C) E. coli BL21(DE3) cells were infected at a multiplicity of 0.5 viruses per cell with either wt T4 (n=6), uvsX mutant (n=5), or gene59 mutant (n=3). Thus, on average, most infected cells contain a single virus. (D) The first 15 minutes of the infection in (C) are plotted. The symbols used in graphs are as follows: wt, open squares; uvsX mutant, open circles; gene59 mutant, open triangles. Error bars indicate standard error.

Figure 3

Relative abundance of size-fractionated nascent T4 DNA synthesized early during infection. Wild type T4D and the recombination uvsX mutant were used to infect E. coli BL21(DE3) cells at a multiplicity of 0.5 viruses per cell. Total viral DNA harvested from infections at 6, 7, and 8 minutes post infection was size fractionated on alkaline agarose gels and used to generate random primed probes as described in Materials and Methods. These labeled DNAs were then used to probe the T4 genomic macroarray, immobilized on nylon membranes, and the relative abundance of viral DNA at each locus along the array was calculated as described in Materials and Methods. (A) Wild type nascent T4D DNA, 3–6 kb. (B) Wild type nascent T4D DNA, 6–10 kb. The filled circles used in (A) and (B) are 6 minutes, black: 7 minutes, orange: 8 minutes, blue. (C) Mutant uvsX nascent DNA, 3–6 kb. (D) Mutant uvsX nascent DNA, 6–10 kb. The open circles used in (C) and (D) are 6 minutes, black: 7 minutes, orange: 8 minutes, blue. The position along the 168 kb T4 genome is identified in kilobases along the x-axis.

Figure 4

T4 DNA replication dynamics in wt and recombination deficient infections. DNA synthesis was monitored across the viral genome using labeled PCR fragments from the T4 macroarray to probe blotted DNA from infections. The amount of viral DNA present at a given time point at a given locus was plotted as a fold increase over the DNA in the same infection at the same locus at 2 minutes post infection, as described in Materials and Methods. (A) E. coli BL21(DE3) cells were infected with wt T4D (n=3) at a multiplicity of 0.5 viruses per cell, where n is the number of independent experiments. (B) The same wt T4D infections at later time points. The filled circles used in (A) and (B) are 7 minutes, orange: 8 minutes, blue: 10 minutes, red; 12 minutes, purple; 15 minutes, green. (C) E. coli BL21(DE3) cells were infected with mutant uvsX (n=4) phage at a multiplicity of 0.5 viruses per cell. (D) The same uvsX infections at later time points. The open circles used in (C) and (D) are 7 minutes, orange: 8 minutes, blue: 10 minutes, red; 12 minutes, purple; 15 minutes, green. The position along the 168 kb T4 genome is identified in kilobases along the x-axis. To maintain graphic clarity only the upper extent of the standard error at each data point is indicated with error bars.

Figure 5

Size distribution of nascent T4 DNA synthesized early during infection. (A) Wild type T4D and the recombination mutant uvsX were used to infect E. coli BL21(DE3) cells at a multiplicity of 0.5 viruses per cell. Total viral DNA, harvested from wt and uvsX infections at 2, 6, 8, 10, 12, 15, and 20 minutes post infection, was size fractionated on an alkaline agarose gel, transferred to nylon and probed with full length T4 DNA as described in Materials and Methods. Numbers above lanes indicate minutes post infection and M identifies the PacI digested viral DNA molecular weight standard. (B) The blot in (A) was exposed to a phosphoimager screen and scanned. To determine the fraction of DNA shorter than 27 kb, the PSL (detected radiation) in a given lane associated with 2.7 to 27 kb DNA was divided by the PSL present in the total DNA in the same lane, from 2.7 kb to the top of the well, as described in Materials and Methods. Results from wt infections are graphed with filled bars and uvsX infections with open bars. (C) The blot in (A) was stripped and reprobed with a PCR fragment that overlaps the putative position of oriE, LP16 from Table 1. The fraction of DNA less than 27 kb was then calculated as in (B).