The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

One of the essential components of a phosphatase that specifically dephosphorylates the Saccharomyces cerevisiae RNA polymerase II (RPII) large subunit C-terminal domain (CTD) is a novel polypeptide encoded by an essential gene termed FCP1. The Fcp1 protein is localized to the nucleus, and it binds the largest subunit of the yeast general transcription factor IIF (Tfg1). In vitro, transcription factor IIF stimulates phosphatase activity in the presence of Fcp1 and a second complementing fraction. Two distinct regions of Fcp1 are capable of binding to Tfg1, but the C-terminal Tfg1 binding domain is dispensable for activity in vivo and in vitro. Sequence comparison reveals that residues 173-357 of Fcp1 correspond to an amino acid motif present in proteins of unknown function predicted in many organisms.Promoter-dependent transcription by RNA polymerase II (RPII) requires six general transcription factors (reviewed in ref.

E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner.

TFIIF (RAP30/74) is a general initiation factor that also increases the rate of elongation by RNA polymerase II. A two-hybrid screen for RAP74-interacting proteins produced cDNAs encoding FCP1a, a novel, ubiquitously expressed human protein that interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74. Related cDNAs encoding FCP1b lack a carboxylterminal RAP74-binding domain of FCP1a. FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the carboxyl-terminal domain of the largest RNA polymerase II subunit. FCP1 is also a stoichiometric component of a human RNA polymerase II holoenzyme complex. Initiation of transcription by RNA polymerase (RNAP)1 II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in Ref. 1). Beginning with TFIID, whose TATA box-binding protein subunit recognizes the TATA box present in many promoters, these factors can assemble in an ordered pathway in vitro onto a promoter (2, 3), resulting in the formation of a preinitiation complex containing more than 40 polypeptides. Subsequently, however, yeast and mammalian RNAP II holoenzymes that contain several or all of the general transcription factors and other polypeptides were discovered (4 -9). There is evidence that transcription by RNAP II in Saccharomyces cerevisiae generally depends on such a holoenzyme (10) and that recruitment of yeast holoenzyme to a promoter would lead to a high rate of transcription (11).During or shortly after initiation by RNAP II, the carboxylterminal domain (CTD) of its largest subunit becomes heavily phosphorylated and remains so during transcript elongation (12). The phosphorylated form of RNAP II is designated RNAP IIO, whereas the unphosphorylated form is designated RNAP IIA. One subunit of TFIIH is a protein kinase that can phosphorylate the CTD (13). Phosphorylation of the CTD by PTEFb, a different Drosophila CTD kinase, has been shown to enhance the processivity of chain elongation by RNAP II in vitro (14). Concomitant with or following the termination of transcription, the CTD must be dephosphorylated by a protein phosphatase, since RNAP IIO cannot assemble directly into a preinitiation complex on either the adenovirus-2 major late or murine dihydrofolate reductase promoter in vitro (15-17). Accordingly, CTD phosphatase may function as a global regulator of gene expression by controlling the pool of RNAP IIA available for initiation. A phosphatase whose activity is stimulated by RAP74 and dephosphorylates the CTD in a processive manner has been purified from HeLa cell extracts (18,19).Certain activator proteins increase the efficiency of RNA chain elongation downstream from the promoter. For example, RNAP II pauses with an unphosphorylated CTD about 25-40 nucleotides downstream from the initiation site of Drosophila hsp70 genes and is stimulated by heat shock and the heat shock factor to become phosphorylated and leave these pause sites (Ref. 20 and references therein). Increasing evidence suppo...

Replication of the papillomavirus genome is initiated by the assembly of a complex between the viral E1 and E2 proteins at the origin. The E1 helicase is comprised of a C-terminal ATPase/helicase domain, a central domain that binds to the origin, and an N-terminal regulatory region that contains nuclear import and export signals mediating its nucleocytoplasmic shuttling. We previously reported that nuclear accumulation of E1 has a deleterious effect on cellular proliferation which can be prevented by its nuclear export. Here we have shown that nuclear accumulation of E1 from different papillomavirus types blocks cell cycle progression in early S phase and triggers the activation of a DNA damage response (DDR) and of the ATM pathway in a manner that requires both the origin-binding and ATPase activities of E1. Complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Transient viral DNA replication still occurs in S-phase-arrested cells but surprisingly is neither affected by nor dependent on induction of a DDR and of the ATM kinase. Finally, we provide evidence that a DDR is also induced in human papillomavirus type 31 (HPV31)-immortalized keratinocytes expressing a mutant E1 protein defective for nuclear export. We propose that nuclear export of E1 prevents cell cycle arrest and the induction of a DDR during the episomal maintenance phase of the viral life cycle and that complex formation with E2 further safeguards undifferentiated cells from undergoing a DDR when E1 is in the nucleus.Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the differentiating epithelium of the skin or mucosa (reviewed in references 10 and 91). About 25 types infect the anogenital tract (6, 19), characterized either as low-risk or high-risk types according to their association with benign or malignant hyperproliferative lesions. Clinically, low-risk HPV types cause benign warts while high-risk types are associated with lesions that can progress to cancer (28,56,63,86).The HPV life cycle is dependent on the differentiation program that keratinocytes undergo within a stratified epithelium. Viral DNA replication is required during the three distinct phases of the viral life cycle (reviewed in references 30 and 36). Upon infection of cells from the basal layer of the epithelium, the viral genome is established as a nuclear episome and is replicated by up to 50 to 100 copies (reviewed in reference 30). These episomes are then maintained at a constant copy number by low levels of replication in the lower layers of the epithelium. During this maintenance phase, viral DNA replication is thought to occur only once per cell cycle, during S phase, and in synchrony with replication of the host DNA (32). Finally, as the infected keratinocytes reach the uppermost differentiated layers of the epithelium, the copy number of the viral episome is amplified to very high levels (reviewed in reference 30), presumably through multiple rounds of replication in S-phase-arrested c...

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.

The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by singlestranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.Papillomaviruses are small DNA viruses that are the etiological agents of benign and malignant lesions of the differentiating mucosal and cutaneous epithelium (reviewed in references 6, 20, 45, and 61). The life cycle of these viruses is closely associated with the differentiation program that occurs in the epithelium. Soon after infection, the viral genome is established as a low-copy-number extrachromosomal episome in the nuclei of infected basal cells. As these infected cells start to differentiate from the basal layer toward the upper portion of the epithelium, the viral episome is replicated and maintained at approximately 50 copies per cell. When the infected cells reach the suprabasal layers, amplification of the viral episome occurs to a high copy number, probably as a result of a change in the mode of DNA replication from a bidirectional theta mode to a rolling-circle mode (11). In these upper layers, capsid proteins are synthesized and viral particles are assembled, and they are e...

Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.Transcription initiation by RNA polymerase II (RNAPII) requires the assembly of a multiprotein complex at the promoter. This complex consists of RNAPII, general transcription factors, and the SRB (suppressor of RNA polymerase B) or mediator proteins which are involved in the positive and negative regulation of transcription. Assembly of this preinitiation complex can be made to occur in a stepwise fashion in vitro (11), but most transcriptional initiation events in Saccharomyces cerevisiae appear to use a preassembled RNAPII holoenzyme containing most of the essential factors (57).TFIIH is a general transcription factor that has an associated helicase, as well as protein kinase activity (26, 27, 52). One of the major targets for phosphorylation by TFIIH in the transcription initiation complex is the unique carboxy-terminal domain (CTD) of the largest subunit of RNAPII. This CTD consists of tandem repeats of the consensus sequence Tyr-SerPro-Thr-Ser-Pro-Ser, which is repeated 52 times in human RNAPII and 26 or 27 times in S. cerevisiae (1,22). Roles for the CTD during transcription initiation, promoter clearance, chain elongation, and transcript processing have been suggested (9, 43). RNAPII molecules with a hypophosphorylated CTD are preferentially recruited to the initiation complex in vitro (18, 40), whereas the elongating RNAPII in vivo usually has a hyperphosphorylated CTD (12, 45). The purified RNAPII holoenzyme contains hypophosphorylated forms of the CTD (37), whereas a purified form of the elongating RNAPII complex has a CTD that is heavily phosphorylated (47). Therefore, it seems that the transcription cycle involves cyclical phosphorylation and dephosphorylation of the RNAPII CTD (24).Gene-specific roles ...