ATP-dependent Unwinding of a Minimal Origin of DNA Replication by the Origin-binding Protein and the Single-strand DNA-binding Protein ICP8 from Herpes Simplex Virus Type I

Abstract

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...

Herpes Virus Replication

“…Thus, UL28 has higher affinity for pac1 sites that have been heat-induced to form a unique structure (94). This high affinity interaction may be similar to that seen at HSV-1 oriS* and UL9 during the initiation of replication (60).…”

“…This process has been demonstrated on a number of oriS containing substrates where unwinding generates long single-stranded stem-loop structures (58,59). This process may proceed via a conformational DNA intermediate, oriS*, that is formed by intrastrand hairpin formation at the origin (60).…”

Functional Properties of the Herpes Simplex Virus Type I Origin-binding Protein Are Controlled by Precise Interactions with the Activated Form of the Origin of DNA Replication

“…Proteins and Nucleic Acids-The UL9 protein, ⌬OBP, and ICP8 were purified as described previously (13,26). Oligonucleotides (Table I) purified by reverse phase-high pressure liquid chromatography were purchased from DNA Technology A/S.…”

“…In the second step, OBP assisted by ICP8 and at the expense of ATP hydrolysis is able to separate the two strands completely and produce an OBP⅐oriS* complex (13). On supercoiled DNA, OBP together with ICP8 catalyzes additional unwinding as seen in experiments using electron microscopy (24).…”

“…Therefore, it is probable that the initiator protein and the replicator sequence co-evolve to ascertain the necessary specificity and efficiency needed for species-specific amplification of viral genomes. To understand in biochemical terms how this is achieved, we have studied the interaction of HSV-1 OBP and the viral origin of replication oriS as well as the interaction of OBP with a probable intermediate in the activation process referred to as oriS* (11)(12)(13). OriS* is an unwound form of double-stranded oriS in which a stable hairpin with a 3Ј-singlestranded region is formed (Fig.…”