The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5 side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.Initiation of replication of viral and cellular chromosomes involves the assembly of multienzyme complexes, replisomes, at unique locations (1). Sequence-specific DNA-binding proteins, initiator proteins, identify the origins of DNA replication and facilitate the conversion of duplex DNA to single-stranded templates for DNA synthesis. The origins of DNA replication display a structural diversity between species that might reflect variations in the mechanisms by which the origins are activated and controlled. In addition, it might also be important to create diversity to ascertain that chromosomes can be uniquely identified and preferentially replicated.We have studied Herpes simplex virus type I to learn more about the molecular mechanisms that underlie initiation of DNA synthesis and subsequent events at the replication fork. HSV-1 1 DNA replication requires seven virally encoded proteins (2, 3). The origin binding protein, OBP, is encoded by the UL9 gene (4, 5). It is a sequence-specific DNA-binding protein and a 3Ј to 5Ј DNA helicase (4, 6, 7). The helicase activity of OBP is specifically stimulated by the viral single-stranded DNA-binding protein ICP8 (8, 9). The putative viral replisome consists of a trimeric helicase-primase composed by the products of the UL5, UL8, and UL52 genes as well as a processive DNA polymerase containing the UL30 and UL42 gene products (2).Initiation of HSV-1 DNA replication is dependent on the viral origins of replication oriS and oriL (3, 10). They are palindromic sequences characterized by two inverted copies of the recognition sequence for OBP, GTTCGCAC (11). The two binding sites are referred to as box I and box II. A third site, box III, does not bind OBP with high affinity despite extensive sequence homology. Box I and box II are separated by a spacer sequence containing 18 alternating AT bas...
The Herpes simplex virus type I origin-binding protein, OBP, is encoded by the UL9 gene. OBP binds the origin of DNA replication, oriS, in a cooperative and sequence-specific manner. OBP is also an ATP-dependent DNA helicase. We have recently shown that singlestranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by OBP. OriS* contains a stable hairpin formed by complementary base pairing between box I and box III in oriS. Here we show that OBP, in the presence of the singlestranded DNA-binding protein ICP8, can convert an 80-base pair double-stranded minimal oriS fragment to oriS* and form an OBP-oriS* complex. The formation of an OBP-oriS* complex requires hydrolysable ATP. We also demonstrate that OBP in the presence of ICP8 and ATP promotes slow but specific and complete unwinding of duplex minimal oriS. The possibility that the OBPoriS* complex may serve as an assembly site for the herpes virus replisome is discussed.DNA replication starts at specific sites on chromosomes referred to as origins of DNA replication. Sequence-specific DNAbinding proteins recognize the origins of replication and facilitate local unwinding of duplex DNA. Once single-stranded DNA has been exposed the remaining replication proteins may assemble into a multienzyme complex frequently referred to as the replisome (1). It is likely that the origins of DNA replication have structural features that contribute to the efficiency and regulation of initiation of DNA replication during one or both of these steps.Initiation of Herpes simplex virus DNA replication depends on two largely homologous origins of replication in the viral genome: oriL and oriS (2-4). There are three copies of the recognition sequence, boxes I, II, and III, for the origin-binding protein, OBP, 1 in oriS, and they are arranged in two palindromes (5). Box I and box III are part of an evolutionarily conserved palindrome that forms a stable hairpin in singlestranded DNA. Box I and box II are separated by an AT-rich spacer sequence that varies in length and nucleotide composition between different ␣ herpes viruses. In oriS from HSV-1 box I, box II and the AT-rich sequence form a 46-bp palindrome. Genetic analyses have demonstrated that the boxes I, II, and III as well as their precise arrangement in oriS affect the efficiency of origin-dependent DNA replication in vivo (5-11).We have previously demonstrated that heat-treated duplex oriS and single-stranded oligonucleotides co-linear with the upper strand of oriS adopt a novel conformation, oriS*, and OBP forms a very stable and specific complex with oriS* (5, 12). OriS* contains a stable hairpin formed by complementary base pairing between box I and box III (5, 12). Biochemical and genetic experiments suggest that this feature is important not only for the formation of the OBP-oriS* complex but also for efficient initiation of DNA replication at oriS (12).The initiator protein, OBP, is a sequence-specific DNA-binding protein and a DNA helicase (4). The DNA helicase activi...
The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III-box I hairpin with a 3 single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III-box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III-box I hairpin in partially unwound DNA.
This study comprehensively investigates the effect of cryogenic nozzle inlet temperature on the flow structure and interactions of an under-expanded supersonic jet with a spherical solid surface. A combined experimental and numerical approach was employed to achieve this goal, utilizing high-speed Z-type schlieren visualization and Reynolds-averaged Navier–Stokes simulations with a Redlich–Kwong real gas equation of state. This study is significant as it addresses a relatively unexplored area of research on the flow structure of the cryogenic under-expanded supersonic jet. The study examines the shock pattern and interaction region through varying static inlet temperature (Tin = 178–290 K) and nozzle pressure ratio (NPR 5–14). Additionally, parameters including nozzle exit-to-throat area ratio (A/A* = 1.277), the distance between the sphere and the nozzle (1.5 cm), and the diameter of the sphere (d = 1.5 cm) were considered fixed. The results show that the supersonic jet exhibits a change in shock patterns in the first shock cell concerning the location and width of the Mach disk, accompanied by a shift in the location of the last shock crossing point and the shock plate. The simulation provides a more detailed insight into the flow, indicating a temperature drop to 105 K in the case of the cryogenic nozzle inlet. At such a low temperature, the compressibility factor exhibits a 5% reduction from unity, while in the case of the ambient nozzle inlet, the minimum temperature at the nozzle exit reached 170 K, leading to only a 1% drop in the compressibility factor, which is negligible. It triggers different flow structures concerning the nozzle inlet temperature. These findings can contribute to the complex flow structures of supersonic jets seen in different industrial and scientific fields.
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