Initiation of bacteriophage T4 DNA replication and replication fork dynamics: a review in the Virology Journal series on bacteriophage T4 and its relatives

Abstract

Bacteriophage T4 initiates DNA replication from specialized structures that form in its genome. Immediately after infection, RNA-DNA hybrids (R-loops) occur on (at least some) replication origins, with the annealed RNA serving as a primer for leading-strand synthesis in one direction. As the infection progresses, replication initiation becomes dependent on recombination proteins in a process called recombination-dependent replication (RDR). RDR occurs when the replication machinery is assembled onto D-loop recombination intermediates, and in this case, the invading 3' DNA end is used as a primer for leading strand synthesis. Over the last 15 years, these two modes of T4 DNA replication initiation have been studied in vivo using a variety of approaches, including replication of plasmids with segments of the T4 genome, analysis of replication intermediates by two-dimensional gel electrophoresis, and genomic approaches that measure DNA copy number as the infection progresses. In addition, biochemical approaches have reconstituted replication from origin R-loop structures and have clarified some detailed roles of both replication and recombination proteins in the process of RDR and related pathways. We will also discuss the parallels between T4 DNA replication modes and similar events in cellular and eukaryotic organelle DNA replication, and close with some current questions of interest concerning the mechanisms of replication, recombination and repair in phage T4.

Figure 1

Location of the T4 origins of replication . The linear 169 kb T4 genome is circularly permuted and has no defined telomeres, so it is depicted in this diagram as a circle. The positions of major T4 origins are indicated with green lollypops. The positions of major T4 open reading frames (>100 amino acids) are indicated with arrows and are color coded to indicate the timing of transcription: blue, early; yellow, middle; and red, late transcripts [5,19]. Three relevant smaller open reading frames are also included: soc near oriA; rI.-1 near oriC; and repEA near oriE.

Figure 2

Two modes of recombination-dependent replication (RDR) . During semi-conservative RDR, primase action on the displaced strand of the D-loop allows lagging strand synthesis (panel A). In bubble-migration synthesis, lagging strand synthesis does not occur, and the newly synthesized single strand is extruded from the back of the D-loop as new DNA is synthesized at the front of the D-loop (panel B). In this and subsequent figures, new leading strand replication is in solid red and new lagging strand replication is in dashed red; the two starting molecules are differentiated by the green versus black colors.

Figure 3

Double-strand break repair models . The SDSA model for DSB repair invokes a limited amount of bubble-migration synthesis using one end of a double-strand break, followed by extrusion of the extended 3' end and capture of the second broken end (panel A). The extensive chromosomal replication (ECR) model invokes two successive rounds of semi-conservative replication (panel B). Depending on which product of the first round of replication is chosen for the second round of strand invasion, the two broken ends of the original double-strand break can end up in different molecules rather than being linked back together again. The final stages of elongation are not shown, but would result in three complete product molecules.

Figure 4

Fork regression model for bypass of leading-strand damage . In this model, leading-strand replication encounters a blocking lesion (solid rectangle) while lagging-strand replication proceeds past the site of the lesion. Replication fork regression leads to an accurate template for extension of the blocked leading-strand product, and re-advancing the fork (reversing the regression) puts the newly synthesized segment of leading-strand product (blue line) opposite the blocking lesion.