DNA Replication Efficiency Depends on Transcription Factor-Binding Sites

Turner, William J.; Woodworth, Mary E.

doi:10.1128/jvi.75.12.5638-5645.2001

Cited by 12 publications

(11 citation statements)

References 70 publications

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“…Whereas the auxiliary transcriptional elements (i.e. the 21-bp repeats and the SV40 enhancer) are not essential for viral DNA replication, they can enhance replication activity and are necessary for the efficient SV40 DNA replication in vivo (33,(37)(38)(39). Interestingly, the SV40 enhancer was found to play a critical role in RAX-mediated enhancement of gene expression (Fig.…”

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confidence: 99%

A Novel Role for RAX, the Cellular Activator of PKR, in Synergistically Stimulating SV40 Large T Antigen-dependent Gene Expression

Yang

Ito²,

May

2003

Journal of Biological Chemistry

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The double-stranded (ds) RNA-binding protein RAX was discovered as a stress-induced cellular activator of the dsRNA-dependent protein kinase (PKR), a key regulator of protein synthesis in response to viral infection and cellular stress. We now report a novel function of RAX, independent of PKR, to enhance SV40 promoter (origin)/enhancer-dependent gene expression. Several mammalian cell lines including COS-7, CV-1, and HeLa cells were tested. Results reveal that the SV40 large T antigen is required for RAX-mediated, synergistic enhancement of gene expression. RAX augments SV40 regulatory element-dependent DNA replication and transcription. The mechanism requires the SV40 enhancer, a viral transcriptional element that is necessary for efficient SV40 DNA replication in vivo. Mutational analysis reveals that the dsRNA-binding domains of RAX are required for the gene expression enhancing function. Thus, in addition to stimulating PKR activity, RAX can positively regulate both SV40 large T antigen-dependent DNA replication and transcription in a mechanism that may alter the interaction of the cellular factor(s) with the SV40 enhancer via the dsRNA-binding domains of RAX. This novel function of RAX may have implications for regulation of mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes.

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confidence: 99%

A Novel Role for RAX, the Cellular Activator of PKR, in Synergistically Stimulating SV40 Large T Antigen-dependent Gene Expression

Yang

Ito²,

May

2003

Journal of Biological Chemistry

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“…Transcriptional events proceed from this large, activated complex (9,42). Both cis-acting regulatory sequences near ori and proteins acting in trans appear to have an important function in controlling replication (4,5,14,29,45,50,61). Bent DNA may facilitate interaction between cis-DNA elements and trans-acting factors by organizing local chromatin infrastructure (44).…”

Section: Containing the Sequences 5ј-(a/t)(a/t)a‫3-ء‬ј And 5ј-(a/t)(gmentioning

confidence: 99%

“…Unwinding of the AT tract is distinct from the denaturing that occurs in the EP region. Because the amount of dimethyl sulfate methylation of internal hydrogen binding sites is only about 5% of that occurring in the EP region, it is apparent that the AT tract is not denaturing but rather untwisting its doublestranded conformation (4, 5).To identify the specific elements that regulate replication efficiency in vivo, our laboratory has used plasmid constructs derived either from SV40 evolutionary variants (6, 58) or from rearranged or duplicated portions of the wild-type SV40 regulatory region (33,34,61). Figure 1 summarizes the organization of the regulatory regions and the effect on the relative replication efficiency (RRE) of some of these SV40 plasmid constructs (34).…”

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confidence: 99%

“…To identify the specific elements that regulate replication efficiency in vivo, our laboratory has used plasmid constructs derived either from SV40 evolutionary variants (6, 58) or from rearranged or duplicated portions of the wild-type SV40 regulatory region (33,34,61). Figure 1 summarizes the organization of the regulatory regions and the effect on the relative replication efficiency (RRE) of some of these SV40 plasmid constructs (34).…”

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Relationship among Location of T-Antigen-Induced DNA Distortion, Auxiliary Sequences, and DNA Replication Efficiency

Okuley

Call

Mitchell

et al. 2003

J Virol

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T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork.Simian virus 40 (SV40) DNA replication has been widely used as a model to study DNA replication in eukaryotes (reviewed in references 7 and 19); the events before the initiation of replication are particularly interesting. SV40 has a welldefined origin of replication (2,10,22,43), and the initiation of replication depends on a single viral protein, T antigen (T-ag) (3,16,36,59). Except for T-ag, all proteins required for DNA replication are supplied by the host cell, and with the development of an in vitro SV40 replication system, their identity and function have been well characterized (35, 65; reviewed in references 7, 30, and 53). The SV40 core ori is a 64-bp region (20,27,43,54) that consists of three functional domains (13, 36, 45): a 17-bp adenine/thymine-rich (AT) region, a central 27-bp palindrome (PEN), and an early palindrome (EP).Located proximal to the AT tract are the promoter and enhancer elements of the SV40 regulatory region. The bidirectional promoter element for SV40 transcription consists of three 21-bp tandem repeats, each containing two copies of the conserved 5Ј-GGGCGG-3Ј (GC box) sequence. The GC boxes are binding sites for the transcription factor Sp1 (16,24,26). The enhancer contains two 72-bp repeats, each with multiple binding sites for transcription factors, including those belonging to the AP family (31).T-ag binds as a trimer to T-ag binding site I (38) located on the early transcription side of ori. The presence of site I facilitates T-ag-dependent unwinding of the DNA (28) and increases the frequency of initiation of DNA replication (25). T-ag binding site II, located at the PEN region in ori, is essential for the initiation of replication (15,17,20,43,46). T-ag forms double hexameric lobes that surround the PEN region (12, 37), and the helicase activity of the hexamers unwinds the DNA bidirectionally (11, 63). T-ag binding also occurs at site III located within the three 21-bp GC-rich repeats on the late transcription side of ori (23,38,60).T-ag binding to ori and flanking sequences induces structural distortions of the region. Both dimethyl sulfate protection and p...

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“…Although the analysis of these sequence elements in either oriLyt function or local transcription regulation has just begun (33), their distribution resembles the organization of some complex transcriptional enhancers, and the existence of these binding sites suggests the presence of putative promoter regions. Transcriptional control elements and enhancers are indeed common components of many viral and eukaryotic origins of DNA replication (21,42,48,50,51). Given the complex structure of HCMV oriLyt, the lack of an apparent transacting factor comparable to the HSV origin-binding protein (41), and the recent identification of intraorigin RNA transcripts that in part form stable and persistent RNA-DNA hybrid structures within oriLyt (45), the mechanism of replication initiation in HCMV may be different from that employed by other herpesviruses.…”

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Analysis of Human Cytomegalovirus ori Lyt Sequence Requirements in the Context of the Viral Genome

Borst

Messerle

2005

J Virol

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During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, oriLyt. Many herpesviruses harbor more than one lytic origin, but so far, only one oriLyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV oriLyt was described as a core region of 1.5 kbp (minimal oriLyt) flanked by auxiliary sequences required for maximal replication activity (complete oriLyt). It remained unclear whether minimal oriLyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of oriLyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete oriLyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete oriLyt was then ectopically reinserted into the oriLyt-depleted genomes. Only the mutant genomes carrying complete oriLyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal oriLyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal oriLyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV oriLyt.Human cytomegalovirus (HCMV), like all herpesviruses, has at least two different modes of infection, either lytic replication or latency. Whereas the replication mode of the HCMV genome during latency is rather elusive, substantial progress has been made in dissecting the molecular steps that occur during lytic DNA replication (35, 39; for a review, see reference 41). Following cell entry, HCMV capsids are transported to the nuclear pores, where they release the linear genomes into the cell nucleus. Then, the linear genomes are rapidly circularized and serve as templates for transcription and replication. It has been proposed that the initial steps in DNA replication of herpesviruses take place by a theta-like mechanism, followed by a rolling-circle mechanism of replication at later stages of the lytic infection cycle (reviewed in references 36 and 46). This view of herpesvirus DNA replication has recently been challenged by the observation that genome circularization seems not to be a prerequisite for replication of herpes simplex virus (HSV) and the suggestion that linear genomes serve as replication templates (31). It remains to be shown whether the same applies to HCMV and other herpesviruses as well.Regardless of which mechanism is used, lytic replication of herpesviruses initiates at defined regions of the viral genomes, th...

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DNA Replication Efficiency Depends on Transcription Factor-Binding Sites

Cited by 12 publications

References 70 publications

A Novel Role for RAX, the Cellular Activator of PKR, in Synergistically Stimulating SV40 Large T Antigen-dependent Gene Expression

A Novel Role for RAX, the Cellular Activator of PKR, in Synergistically Stimulating SV40 Large T Antigen-dependent Gene Expression

Relationship among Location of T-Antigen-Induced DNA Distortion, Auxiliary Sequences, and DNA Replication Efficiency

Analysis of Human Cytomegalovirus ori Lyt Sequence Requirements in the Context of the Viral Genome

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