End Processing Activities Research Articles

To live or to die: a matter of processing damaged DNA termini in neurons

Defects in the repair of deoxyribonucleic acid (DNA) damage underpin several hereditary neurological diseases in humans. Of the different activities that repair chromosomal DNA breaks, defects in resolving damaged DNA termini are among the most common causes of neuronal cell death. Here, the molecular mechanisms of some of the DNA end processing activities are reviewed and the association with human neurodegenerative disease is discussed.

GCN5-dependent acetylation of HIV-1 integrase enhances viral integration

BackgroundAn essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300.ResultsIn this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273) are targeted by GCN5 acetylation, three of which (K264, K266, and K273) are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258) does not affect this process.ConclusionsThe results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.

The DNA binding and 3′-end preferential activity of human tyrosyl-DNA phosphodiesterase

Human tyrosyl-DNA phosphodiesterase (Tdp1) processes 3′-blocking lesions, predominantly 3′-phosphotyrosyl bonds resulting from the trapping of topoisomerase I (Top1) cleavage complexes. The controversial ability of yeast Tdp1 to hydrolyze 5′-phosphotyrosyl linkage between topoisomerase II (Top2) and DNA raises the question whether human Tdp1 possesses 5′-end processing activity. Here we characterize the end-binding and cleavage preference of human Tdp1 using single-stranded 5′- and 3′-fluorescein-labeled oligonucleotides. We establish 3′-fluorescein as an efficient surrogate substrate for human Tdp1, provided it is attached to the DNA by a phosphodiester (but not a phosphorothioate) linkage. We demonstrate that human Tdp1 lacks the ability to hydrolyze a phosphodiester linked 5′-fluorescein. Using both fluorescence anisotropy and time-resolved fluorescence quenching techniques, we also show the preferential binding of human Tdp1 to the 3′-end. However, DNA binding competition experiments indicate that human Tdp1 binding is dependent on DNA length rather than number of DNA ends. Lastly, using surface plasmon resonance, we show that human Tdp1 selectively binds the 3′-end of DNA. Together, our results suggest human Tdp1 may act using a scanning mechanism, in which Tdp1 bind non-specifically upstream of a 3′-blocking lesion and is preferentially stabilized at 3′-DNA ends corresponding to its site of action.

Functions and Regulation of Artemis: A Goddess in the Maintenance of Genome Integrity

Artemis is a structure-specific endonuclease when associated with and phosphorylated by DNA-dependent protein kinase catalytic subunit. This structure-specific endonuclease is responsible for the resolution of hairpin coding ends in V(D)J recombination. In DNA double-strand break repair, Artemis is implicated in the end-processing step of the non-homologous end-joining (NHEJ) pathway. Recently, we have demonstrated that the involvement of Artemis in NHEJ depends on the type of DNA damage. Interestingly, recent evidence suggests that the end-processing activity is not the only function of Artemis. Indeed, Artemis is rapidly phosphorylated by ataxia telangiectasia mutated in response to DNA damage, and such phosphorylation of Artemis appears to be involved in the regulation of cell cycle checkpoints. These findings suggest that Artemis is a multifunctional protein participating in the maintenance of genome integrity at two distinct levels; one at the end processing step of NHEJ, and the other at the signaling pathway of cell cycle regulation. Therefore, understanding Artemis function may give us profound insights into the DNA repair network. In this review, we summarize the functions and regulation of Artemis.

DNA damage signalling prevents deleterious telomere addition at DNA breaks

The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage, to increase the accuracy of repair. Here we report that telomerase action is regulated as a part of the cellular response to a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity.

Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT(1)-BRCT(2) domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

Roles for NBS1 in Alternative Nonhomologous End-Joining of V(D)J Recombination Intermediates

Recent work has highlighted the importance of alternative, error-prone mechanisms for joining DNA double-strand breaks (DSBs) in mammalian cells. These noncanonical, nonhomologous end-joining (NHEJ) pathways threaten genomic stability but remain poorly characterized. The RAG postcleavage complex normally prevents V(D)J recombination-associated DSBs from accessing alternative NHEJ. Because the MRE11/RAD50/NBS1 complex localizes to RAG-mediated DSBs and possesses DNA end tethering, processing, and joining activities, we asked whether it plays a role in the mechanism of alternative NHEJ or participates in regulating access of DSBs to alternative repair pathways. We find that NBS1 is required for alternative NHEJ of hairpin coding ends, suppresses alternative NHEJ of signal ends, and promotes proper resolution of inversional recombination intermediates. These data demonstrate that the MRE11 complex functions at two distinct levels, regulating repair pathway choice (likely through enhancing the stability of DNA end complexes) and participating in alternative NHEJ of coding ends.

Long Patch Base Excision Repair in Mammalian Mitochondrial Genomes

The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.

Expression and characterization of the integrase of bovine immunodeficiency virus

In the pol gene of bovine immunodeficiency virus (BIV) there is a sequence, located between the reverse-transcriptase and integrase (IN)-encoding sequences, that is not found in HIV-1 pol gene and encodes a 74-residue polypeptide with homology to dUTPases. We have expressed two BIV IN versions that differ in their amino termini. The longer version, containing the 74-residue sequence, did not show any detectable 3′-end processing and strand transfer IN activities and performed only the IN-associated disintegration. Consequently, the shorter version, lacking the dUTPase-related residues, performed all three activities and is most likely similar to the viral enzyme. A comparison between BIV IN and the well-studied HIV-1 IN, with substrates that mimic the U5 LTR sequences of BIV, HIV-1 and another bovine lentivirus, Jembrana disease virus, revealed that the extra 3′-end sequence beyond the conserved “CA” is probably less important for IN activities than the sequence upstream to the “CA".

Interaction between HIV-1 Rev and Integrase Proteins

Human immunodeficiency virus 1 (HIV-1) Rev and integrase (IN) proteins are required within the nuclei of infected cells in the late and early phases of the viral replication cycle, respectively. Here we show using various biochemical methods, that these two proteins interact with each other in vitro and in vivo. Peptide mapping and fluorescence anisotropy showed that IN binds residues 1-30 and 49-74 of Rev. Following this observation, we identified two short Rev-derived peptides that inhibit the 3'-end processing and strand-transfer enzymatic activities of IN in vitro. The peptides bound IN in vitro, penetrated into cultured cells, and significantly inhibited HIV-1 in multinuclear activation of a galactosidase indicator (MAGI) and lymphoid cultured cells. Real time PCR analysis revealed that the inhibition of HIV-1 multiplication is due to inhibition of the catalytic activity of the viral IN. The present work describes novel anti-HIV-1 lead peptides that inhibit viral replication in cultured cells by blocking DNA integration in vivo.

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3′ untranslated regions

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3′ untranslated region (3′UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3′UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3′UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca2+-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3′-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (Bmax) 0.25 time for petD 3′UTR, and three times for psbA 3′UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that Bmax, not Kd, might be a more important factor in the P-24RNP-3′UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

Further screening of Venda medicinal plants for activity against HIV type 1 reverse transcriptase and integrase

The use of medicinal plants for AIDS-related conditions is common in South Africa. In order to establish an antiviral rationale for the use of these plants we screened fractions of the methanol extracts of medicinal plants for activity against HIV-1 reverse transcriptase (RT) and integrase (IN). The n-butanol fraction obtained from the crude methanol extracts of the roots of Bridelia micrantha (Hochst) Baill. (Euphorbiaceae) was observed to be as the most active inhibiting the RNA-dependent-DNA polymerization (RDDP) activity of HIV-1 RT with an IC50 of 7.3 �g/ml. However, it had no activity on the 3’-end processing activity of HIV integrase. Bioassay-guided fractionation of the n-butanol fraction yielded friedelin and �-sistosterol, which did not inhibit the RDDP of RT or 3’-end processing functions of IN even at a concentration of 500 �M. An uncharacterized fraction obtained in the bioassay-guided fractionating process inhibited the RDDP with an IC50 of 9.6 �g/ml, but had no inhibition on IN. Phytochemical screening indicated the presence of flavonoids and tannins in the uncharacterized fraction.

Design, synthesis, and biological evaluation of chicoric acid analogs as inhibitors of HIV-1 integrase

A series of analogs of the potent HIV-1 integrase (HIV IN) inhibitor chicoric acid (CA) was designed with the intention of ameliorating some of the parent natural product’s undesirable properties, in particular its toxicity, instability, and poor membrane permeability. More than 70 analogs were synthesized and assayed for three types of activity: (1) the ability to inhibit 3′-end processing and strand transfer reactions using recombinant HIV IN in vitro, (2) toxicity against the CD4+ lymphoblastoid cell line, MT2, and (3) anti-HIV activity against HIV LAI. CA analogs lacking one of the carboxyl groups of CA and with 3,4,5-trihydroxycinnamoyl sidechains in place of the caffeoyl group of CA exhibited the most potent inhibition of HIV replication and end-processing activity. Galloyl-substituted derivatives also displayed very potent in vitro and in vivo activities, in most cases exceeding the inhibitory effects of CA itself. Conversely, analogous monocarboxy caffeoyl analogs exhibited only modest inhibition, while the corresponding 3,4-dihydroxybenzoyl-substituted compounds were devoid of activity.

Preliminary Mapping of a Putative Inhibitor-Binding Pocket for Human Immunodeficiency Virus Type 1 Integrase Inhibitors

Molecular modeling studies have identified a putative human immunodeficiency virus (HIV) integrase (IN) inhibitor-binding pocket for l-chicoric acid (l-CA) and other inhibitors of IN (C. A. Sotriffer, H. Ni, and A. McCammon, J. Med. Chem. 43:4109-4117, 2000). By using site-directed mutagenesis of several amino acid residues identified by modeling studies, a common inhibitor-binding pocket on IN was confirmed for l-CA and the diketo acid L-731,988. Specifically, the single mutations E92K, Q148A, K156A, K156R, G140S, and G149S, as well as the double mutations C65S-K156N and H67D-G140A were evaluated for their effects on enzymatic activity and inhibitor susceptibility. Each recombinant IN was attenuated for 3'-end processing and strand transfer activities. Most proteins were also attenuated for disintegration; the IN that contained K156R and C65S-K156N, however, displayed disintegration activity similar to that of IN from HIV(NL4-3). All mutant IN proteins demonstrated decreased susceptibility to l-CA, while all mutant proteins except E92K and K156R demonstrated resistance to L-731,988. These data validate the computer modeling data and demonstrate that l-CA and L-731,988 share an overlapping inhibitor-binding pocket that involves amino acids Q148, C65, and H67. The resistance studies confirm that L-731,988 fills one-half of the inhibitor-binding pocket and binds to Q148 but excludes E92, while l-CA fills the entire binding groove and thus interacts with E92. These results provide "wet laboratory" evidence that molecular models of the HIV IN inhibitor-binding pocket can be used for drug discovery.

Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase, RNase H, and Integrase Activities by Hydroxytropolones

Human immunodeficiency virus type I reverse transcriptase (RT) possesses distinct DNA polymerase and RNase H sites, whereas integrase (IN) uses the same active site to perform 3'-end processing and strand transfer of the proviral DNA. These four enzymatic activities are essential for viral replication and require metal ions. Two Mg2+ ions are present in the RT polymerase site, and one or two Mg2+ ions are required for the catalytic activities of RNase H and IN. We tested the possibility of inhibition of the RT polymerase and RNase H as well as the IN 3'-end processing and transfer activities of purified enzymes by a series of 3,7-dihydroxytropolones designed to target two Mg2+ ions separated by approximately 3.7 angstroms. The RT polymerase and IN 3' processing and strand transfer activities were inhibited at submicromolar concentrations, while the RNase H activity was inhibited in the low micromolar range. In all cases, the lack of inhibition by tropolones and O-methylated 3,7-dihydroxytropolones was consistent with the active molecules binding the metal ions in the active site. In addition, inhibition of the DNA polymerase activity was shown to depend on the Mg2+ concentration. Furthermore, selective inhibitors were identified for several of the activities tested, leaving some potential for design of improved inhibitors. However, all tested compounds exhibited cellular toxicity that presently limits their applications.

Rat1p and Rai1p function with the nuclear exosome in the processing and degradation of rRNA precursors

Exoribonucleases function in the processing and degradation of a variety of RNAs in all organisms. These enzymes play a particularly important role in the maturation of rRNAs and in a quality-control pathway that degrades rRNA precursors upon inhibition of ribosome biogenesis. Strains with defects in 3'-5' exoribonucleolytic components of the RNA processing exosome accumulate polyadenylated precursor rRNAs that also arise in strains with ribosome biogenesis defects. These findings suggested that polyadenylation might target pre-rRNAs for degradation by the exosome. Here we report experiments that indicate a role for the 5'-3' exoribonuclease Rat1p and its associated protein Rai1p in the degradation of poly(A)(+) pre-rRNAs. Depletion of Rat1p enhances the amount of poly(A)(+) pre-rRNA that accumulates in strains deleted for the exosome subunit Rrp6p and decreases their 5' heterogeneity. Deletion of RAI1 results in the accumulation of poly(A)(+) pre-rRNAs, and inhibits Rat1p-dependent 5'-end processing and Rrp6p-dependent 3'-end processing of 5.8S rRNA. RAT1 and RAI1 mutations cause synergistic growth defects in the presence of rrp6-Delta, consistent with the interdependence of 5'-end and 3'-end processing pathways. These findings suggest that Rai1p may coordinate the 5'-end and 3'-end processing and degradation activities of Rat1p and the nuclear exosome.

Evaluation of selected South African medicinal plants for inhibitory properties against human immunodeficiency virus type 1 reverse transcriptase and integrase

Seventeen aqueous and methanol extracts from nine South African medicinal plants, ethnobotanically selected, were screened for inhibitory properties against HIV-1 reverse transcriptase (RT). Isolated compounds were additionally evaluated on HIV-1 integrase (IN). The strongest inhibition against the RNA-dependent-DNA polymerase (RDDP) activity of RT was observed with the methanol extract of the stem-bark of Peltophorum africanum Sond. (Fabaceae) (IC 50 3.5 μg/ml), while the methanol extract of the roots of Combretum molle R.Br. ex G. Don (Combretaceae) was the most inhibitory on the ribonuclease H (RNase H) activity (IC 50 9.7 μg/ml). The known compounds bergenin and catechin, and a red coloured gallotannin composed of meta-depside chains of gallic and protocatechuic acids esterified to a 1- O-isobutyroly-β- d-glucopyranose core, were isolated from the methanol extract of the roots and stem-bark of Peltophorum africanum. The gallotannin inhibited the RDDP and RNase H functions of RT with IC 50 values of 6.0 and 5.0 μM, respectively, and abolished the 3′-end processing activity of IN at 100 μM. Catechin showed no effect on RT but had a moderate activity on HIV-1 IN. Bergenin was inactive on both enzymes. The aqueous and methanol extracts were non-toxic in a HeLaP4 cell line at a concentration of 400 μg/ml.

Full-length RAG-2, and Not Full-length RAG-1, Specifically Suppresses RAG-mediated Transposition but Not Hybrid Joint Formation or Disintegration

RAG-1 and RAG-2 initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences flanking a pair of antigen receptor gene segments. Occasionally, the RAG proteins mediate two other alternative DNA rearrangements in vivo: the rejoining of signal and coding ends and the transposition of signal ends into unrelated DNA. In contrast, truncated, catalytically active "core" RAG proteins readily catalyze these reactions in vitro, suggesting that full-length RAG proteins directly or indirectly suppress these undesired reactions in vivo. To discriminate between direct and indirect suppression models, full-length RAG proteins were purified and characterized in vitro. From mammalian cells, full-length RAG-1 is readily purified with core RAG-2 but not full-length RAG-2 and vice versa. Despite differences in DNA binding activity, recombinase containing either core or full-length RAG-1 or RAG-2 possess comparable cleavage, rejoining, and end-processing activity, as well as similar usage preferences for canonical versus cryptic recombination signals. However, recombinase containing full-length RAG-2, but not full-length RAG-1, exhibits dramatically reduced transposition activity in vitro. These data suggest RAG-mediated transposition and rejoining are differentially regulated by the full-length RAG proteins in vivo (the former directly by RAG-2 and the latter indirectly through other factors) and argue that noncore portions of the RAG proteins have little or no direct influence over V(D)J recombinase site specificity.

Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination.

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre-mRNA 3' end processing, binds to the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. We show that the conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3' end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3' end processing activity of Pcf11p and a deficiency of Pcf11p in 3' end processing did not prevent CTD binding. Transcriptional run-on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. We conclude that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination.