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Protozoan parasites undergo complex life cycles that depend on regulated gene expression. However, limited studies on gene regulation in these parasites have repeatedly shown characteristics different from other eukaryotes. Within the Apicomplexa family, little is known about the mechanism of gene expression and regulation in Plasmodium spp. We have been investigating the cis-elements that control basal expression of a sexual stage specific gene in Plasmodium gallinaceum. Previously, we identified by 5' deletion analysis of a reporter construct that the 333bp upstream of the translational start site of pgs28 is sufficient for basal expression, and that the sequence between -333 and 316bp is necessary for such expression. In this report, we identified by linker scanning mutagenesis an 8-bp sequence that is essential for pgs28 transgene expression. This sequence is a target of sequence-specific nuclear factors. Primer extension studies demonstrate that, interestingly, the endogenous pgs28 transcript has two 5' ends, at -65 and +1. We suggest that this 8-bp sequence, CAGACAGC that is situated at +24 to +31 (with respect to the proximal start site), is a novel downstream promoter element in P. gallinaceum that appears to function independently of a TATA box or an Inr element.

During DNA replication, long-chain DNA synthesis on the leading strand requires processivity factors to overcome the tendency of DNA polymerase to dissociate from the template after each catalytic step. In most of the organisms studied to date, these processivity factors interact with the catalytic subunit of polymerase and with DNA to increase the time of association of the holoenzyme with the DNA template. The best-characterized processivity factors are the so-called “sliding clamps,” which include the Escherichia coli β subunit of DNA polymerase III, bacteriophage T4 gp45, and eukaryotic PCNA. Biochemical and crystallographic studies have shown that these factors do not bind directly to DNA but, rather, form multimeric rings around DNA, which permits them to slide along the template (15, 19, 30, 31, 47, 58). Under physiological conditions, the association of a sliding clamp with DNA and its cognate polymerase requires auxiliary proteins that serve as “clamp loaders” (27, 32, 33, 40, 50) and occurs at the primer-template junction (reviewed in reference 20, 27, 44, and 45). A second type of processivity factor is exemplified by thioredoxin, which, unlike the sliding clamps, associates with T7 DNA polymerase in the absence of other protein factors or ATP to form a heterodimer (22, 24, 39, 48). However, like sliding clamps, thioredoxin does not bind DNA directly (39). Thus, during DNA synthesis, the interactions of the sliding clamps and thioredoxin with DNA and their translocation appear to be passive and dependent on their association with their cognate polymerases. For herpes simplex virus (HSV), the details of how processive DNA synthesis occurs have yet to be resolved. The replicative DNA polymerase consists of a heterodimer of two proteins (6, 14). One of these proteins is the catalytic subunit, Pol (also known as UL30), whose intrinsic activities include a 5′-3′ polymerase and a 3′-5′ exonuclease (11, 16, 17, 29, 35, 42, 56). To carry out these functions, Pol has double-stranded DNA (dsDNA)- and single-stranded DNA (ssDNA)-binding activities that also can be found within individual proteolytic fragments of the enzyme (56). Moreover, DNA binding causes a conformational change in Pol (57). However, this DNA binding is not sufficient for highly processive DNA synthesis (14, 21). The second HSV polymerase subunit is UL42, which is a processivity factor (14). It differs from other established processivity factors in that it binds directly and stably to dsDNA, albeit without sequence specificity (36, 43). Unlike sliding clamps, the association of Pol and UL42 does not require additional factors and can occur in the absence of DNA. Both the DNA-binding and Pol-binding activities of UL42 appear to be required for its function as a processivity factor, since mutations that specifically affect either of these activities severely reduce long-chain DNA synthesis and in vivo replication (3, 10). Moreover, the affinity of Pol/UL42 for a hairpin primer-template is greater than that of Pol alone, and the footprint on the dsDNA region of this template is extended when UL42 is present (13). Thus, the available data support the hypothesis (14) that UL42 functions as a tether between Pol and DNA and that its DNA-binding activity is crucial for its function as a processivity factor. However, many of the specifics regarding how UL42 affects the interaction of polymerase with DNA are not known, nor is it known whether UL42, which has high affinity for DNA, affects the movement of the polymerase along the template. In this study, we tested three hypotheses: (i) that UL42 increases the specificity of the holoenzyme for a primer-template configuration, (ii) that UL42 limits the rate of dissociation of the holoenzyme from the primer-template, and (iii) that the increase in processivity conferred by UL42 is achieved at the expense of a decrease in the rate of elongation, i.e., that the polymerase sacrifices speed for distance. To examine these hypotheses, bandshift and competition assays were used to determine the binding affinities of Pol, UL42, and the Pol/UL42 heterodimer on different templates in the presence or absence of magnesium. These assays allowed a comparison of the binding preferences of these proteins to different configurations of DNA. The association and dissociation rates of Pol and Pol/UL42 on DNA in a primer-template configuration were determined. Finally, the rates of elongation of Pol and Pol/UL42 were compared by using a rapid-quench technique. The results indicate that UL42 both increases the specificity of polymerase binding to primer-template DNA and decreases the rate of dissociation from primer-template DNA. However, despite the increased binding to DNA, UL42 does not retard the rate of elongation of polymerase.

The herpes simplex virus DNA polymerase is a heterodimer consisting of a catalytic subunit and the protein UL42, which functions as a processivity factor. It has been hypothesized that UL42 tethers the catalytic subunit to the DNA template by virtue of DNA binding activity (J. Gottlieb, A. I. Marcy, D. M. Coen, and M. D. Challberg, J. Virol. 64:5976-5987, 1990). Relevant to this hypothesis, we identified two linker insertion mutants of UL42 that were unable to bind to a double-stranded-DNA-cellulose column but retained their ability to bind the catalytic subunit. These mutants were severely impaired in the stimulation of long-chain-DNA synthesis by the catalytic subunit in vitro. In transfected cells, the expressed mutant proteins localized to the nucleus but were nonetheless deficient in complementing the growth of a UL42 null virus. Thus, unlike many other processivity factors, UL42 appears to require an intrinsic DNA binding activity for its function both in vitro and in infected cells. Possible mechanisms for the activity of UL42 and its potential as a drug target are discussed.

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 μM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.