Radiolabeled probes were generated by using a PCR fragment and the Random-Primed DNA Labeling kit (Roche). and a Holliday junction. Each of these DNA elements serves as a target for processing by the T4 ATPase/exonuclease complex [gene product (gp) 46/47] and Holliday junction-cleaving enzyme (EndoVII), respectively. In the absence of both gp46 and EndoVII, regressed origin forks are stabilized and persist throughout contamination. In the presence of EndoVII, but not gp46, there is significantly less regressed origin fork accumulation apparently due to cleavage of the regressed fork Holliday junction. In the presence of gp46, but not EndoVII, regressed origin fork DSEs are processed by degradation of the DSE and a pathway that includes recombination proteins. Although both mechanisms can occur independently, they may normally function together as a single fork reactivation pathway. occurs bidirectionally through a two-step process (Fig. 1) (2). A middle-mode promoter and DNA unwinding element promote the formation of an R loop that initiates the first replication fork (3). The mechanism of T4 replication from an R loop has been analyzed (4). In the beginning, the branch structure of the R loop supports the binding of the replicative helicase loader [gene product (gp) 59], which promotes removal of the ssDNA-binding protein (gp32) in favor of the replicative helicase (gp41). Gp59 also functions as a gatekeeper, preventing uncoupled leading-strand extension until gp41 is usually loaded (5, 6). When gp59 is usually absent, replication proceeds at a slower rate before assembly of the replicative helicase, but the helicase (and the associated primase) does weight onto the fork at a reduced efficiency (4). In either case (presence or absence of gp59), when the initial replication fork leaves the origin region, a site-specific branch is usually left behind at the origin; we refer to this transient fork-shaped intermediate as the origin fork (Fig. 1, gray box). After some delay, the origin fork is converted into a functional retrograde replication fork by unknown mechanisms. Presumably, gp59 could weight gp41 directly onto fork intermediates to allow activation without fork processing. However, the delayed activation of the origin fork suggests that other mechanisms also are involved. We propose that the origin fork is a useful model for studying the mechanisms of stalled fork activation in a site-specific manner without the need for replication inhibitors or DNA damage (which typically are used MK-571 to MK-571 generate such models transcripts. Horizontal arrows show the direction of an active replication fork. The highlighted branched molecule is the origin fork, activation of which (last step) is the subject of this study. Bacteriophage T4 replication is usually tightly coupled to recombination. Although replication at early occasions of infection is usually origin-dependent, late replication is driven by recombination proteins in a process called recombination-dependent replication (RDR) (7). Gp46/47, a member of the Rad50/Mre11 family, acts on dsDNA ends (DSEs) that arise during T4 contamination, promoting end degradation and preparing ends for homologous recombination (8, 9). In this regard, gp46/47 exhibits specific proteinCprotein interactions with the T4 recombination mediator protein, UvsY (10). This conversation is thought to recruit UvsY to newly generated ssDNA and to promote loading and filamentation of the T4 strand-exchange protein, UvsX. The invasion of MK-571 a processed DSE into duplex DNA creates a D-loop structure, which, like the R loops discussed in the previous paragraph, can be used to initiate replication. During RDR, the invading 3 end serves as the primer for leading-strand replication, whereas the displaced ssDNA region facilitates gp59-mediated loading of gp41 MK-571 (11). The T4 Holliday junction-cleaving enzyme (EndoVII), the product of gene (21) exhibited that this 2D gel-migration Rabbit Polyclonal to CtBP1 pattern of simple fork regression does not match the observed cone shape. In addition, several different types of replication intermediates also have been shown to migrate within the cone region (5, 19, 22). Using 2D agarose gel electrophoresis, we statement here that this T4 origin fork undergoes fork regression and that regression supports two separate mechanisms of origin fork processing. Regression occurs when the newly created leading and lagging strands of a fork become dissociated from their themes and hybridize to each other (23). This reaction creates a new DSE in the extruded DNA and a Holliday junction at MK-571 the fork. We find that these two DNA elements serve as targets for processing by gp46/47 and EndoVII, respectively. In addition, we describe the appearance of a modified cone region that results from gp46/47-mediated degradation of the regressed origin fork DSE, explaining how regression can contribute to the formation of a cone region much like those explained previously (18C20). Results T4.