Structure of Telomerase with Telomeric DNA.

Telomerase is an RNA-protein complex (RNP) that extends telomeric DNA at the 3' ends of chromosomes using its telomerase reverse transcriptase (TERT) and integral template-containing telomerase RNA (TER). Its activity is a critical determinant of human health, affecting aging, cancer, and stem cell renewal. Lack of atomic models of telomerase, particularly one with DNA bound, has limited our mechanistic understanding of telomeric DNA repeat synthesis. We report the 4.8 Å resolution cryoelectron microscopy structure of active Tetrahymena telomerase bound to telomeric DNA. The catalytic core is an intricately interlocked structure of TERT and TER, including a previously structurally uncharacterized TERT domain that interacts with the TEN domain to physically enclose TER and regulate activity. This complete structure of a telomerase catalytic core and its interactions with telomeric DNA from the template to telomere-interacting p50-TEB complex provides unanticipated insights into telomerase assembly and catalytic cycle and a new paradigm for a reverse transcriptase RNP.

Structural insights into the assembly and mechanism of mpox virus DNA polymerase complex F8-A22-E4-H5.

The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.

MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses.

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in transcription of heat shock protein (HSP)-encoding genes in fly ovaries, but the mechanisms remain unclear. Here, we demonstrate that, in mammalian cells, MRTFs are dispensable for gene induction of HSP-encoding genes. However, the widely used small-molecule inhibitors of the MRTF-SRF transcription pathway, derived from CCG-1423, also efficiently inhibit gene transcription of HSP-encoding genes in both fly and mammalian cells in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF-SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head-bound state.

KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. However, structural features underlying this function are unclear. Here, we determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine-rich insertion in loop-12 termed the 'K-loop', which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native KIF1A loop-12 with that of kinesin-1 resulted in a 6-fold decrease in run length, whereas adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell time assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant than WT. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing binding of the motor in its vulnerable one-head-bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that KIF1A processivity is linearly dependent on the charge of loop-12, further highlighting how loop-12 contributes to the function of this key motor protein.

Ensembles of human myosin-19 bound to calmodulin and regulatory light chain RLC12B drive multimicron transport.

Myosin-19 (Myo19) controls the size, morphology, and distribution of mitochondria, but the underlying role of Myo19 motor activity is unknown. Complicating mechanistic in vitro studies, the identity of the light chains (LCs) of Myo19 remains unsettled. Here, we show by coimmunoprecipitation, reconstitution, and proteomics that the three IQ motifs of human Myo19 expressed in Expi293 human cells bind regulatory light chain (RLC12B) and calmodulin (CaM). We demonstrate that overexpression of Myo19 in HeLa cells enhances the recruitment of both Myo19 and RLC12B to mitochondria, suggesting cellular association of RLC12B with the motor. Further experiments revealed that RLC12B binds IQ2 and is flanked by two CaM molecules. In vitro, we observed that the maximal speed (∼350 nm/s) occurs when Myo19 is supplemented with CaM, but not RLC12B, suggesting maximal motility requires binding of CaM to IQ-1 and IQ-3. The addition of calcium slowed actin gliding (∼200 nm/s) without an apparent effect on CaM affinity. Furthermore, we show that small ensembles of Myo19 motors attached to quantum dots can undergo processive runs over several microns, and that calcium reduces the attachment frequency and run length of Myo19. Together, our data are consistent with a model where a few single-headed Myo19 molecules attached to a mitochondrion can sustain prolonged motile associations with actin in a CaM- and calcium-dependent manner. Based on these properties, we propose that Myo19 can function in mitochondria transport along actin filaments, tension generation on multiple randomly oriented filaments, and/or pushing against branched actin networks assembled near the membrane surface.

Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA.

Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5'→3' direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA-binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA.

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1.

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4ß,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, with koff rates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3ß-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14α C-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.

Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties.

A stop or go switch: glycogen synthase kinase 3ß phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules.

It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previously, we found that GSK3ß plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3ß in vivo. The GSK3ß-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3ß phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3ß non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, we propose that GSK3ß phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.

Inhibiting KSHV replication by targeting the essential activities of KSHV processivity protein, PF-8.

Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are no cures for KSHV infection. KSHV establishes biphasic lifelong infections. During the lytic phase, new genomes are replicated by seven viral DNA replication proteins. The processivity factor's (PF-8) functions to tether DNA polymerase to DNA, so new viral genomes are efficiently synthesized. PF-8 self-associates, interacts with KSHV DNA replication proteins and the viral DNA. Inhibition of viral DNA replication would diminish the infection within a host and reduce transmission to new individuals. In this review we summarize PF-8 molecular and structural studies, detail the essential protein-protein and nucleic acid interactions needed for efficient lytic DNA replication, identify future areas for investigation and propose PF-8 as a promising antiviral target. Additionally, we discuss similarities that the processivity factor from Epstein-Barr virus shares with PF-8, which could promote a pan-herpesvirus antiviral therapeutic targeting strategy.

Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product.

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 µM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 µM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 µM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

Modeling study of kinesin-13 MCAK microtubule depolymerase.

Mitotic centromere-associated kinesin (MCAK) motor protein is a typical member of the kinesin-13 family, which can depolymerize microtubules from both plus and minus ends. A critical issue for the MCAK motor is how it performs the depolymerase activity. To address the issue, the pathway of the MCAK motor moving on microtubules and depolymerizing the microtubules is presented here. On the basis of the pathway, the dynamics of both the wild-type and mutant MCAK motors is studied theoretically, which include the full-length MCAK, the full-length MCAK with mutations in the α4-helix of the motor domain, the mutant full-length MCAK with a neutralized neck, the monomeric MCAK and the mutant monomeric MCAK with a neutralized neck. The studies show that a single dimeric MCAK motor can depolymerize microtubules in a processive manner, with either one tubulin or two tubulins being removed per times. The theoretical results are in agreement with the available experimental data. Moreover, predicted results are provided.

Strategies and procedures to generate chimeric DNA polymerases for improved applications.

Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. KEY POINTS: • Chimeric DNA polymerase is generated by rational design method. • Strategies include domain exchange and addition of proteins, domains, and motifs. • Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment.

The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214-218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

Updates on Mechanisms of Cytochrome P450 Catalysis of Complex Steroid Oxidations.

Cytochrome P450 (P450) enzymes dominate steroid metabolism. In general, the simple C-hydroxylation reactions are mechanistically straightforward and are generally agreed to involve a perferryl oxygen species (formally FeO3+). Several of the steroid transformations are more complex and involve C-C bond scission. We initiated mechanistic studies with several of these (i.e., 11A1, 17A1, 19A1, and 51A1) and have now established that the dominant modes of catalysis for P450s 19A1 and 51A1 involve a ferric peroxide anion (i.e., Fe3+O2¯) instead of a perferryl ion complex (FeO3+), as demonstrated with 18O incorporation studies. P450 17A1 is less clear. The indicated P450 reactions all involve sequential oxidations, and we have explored the processivity of these multi-step reactions. P450 19A1 is distributive, i.e., intermediate products dissociate and reassociate, but P450s 11A1 and 51A1 are highly processive. P450 17A1 shows intermediate processivity, as expected from the release of 17-hydroxysteroids for the biosynthesis of key molecules, and P450 19A1 is very distributive. P450 11B2 catalyzes a processive multi-step oxidation process with the complexity of a chemical closure of an intermediate to a locked lactol form.

Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single DNA Lesions.

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' → 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.

Formation and Effects of Upstream DNA-RNA Base Pairing in Telomerase.

Telomere elongation by telomerase consists of two types of translocation: duplex translocation during each repeat synthesis and template translocation at the end of repeat synthesis. Our replica exchange molecular dynamics simulations show that in addition to the Watson-Crick interactions in the active site, templating RNA can also form base pairs with the upstream regions of DNA, mostly with the second upstream DNA repeat with respect to the 3'-end. At the end of the repeat synthesis, dG10-P442 and dG11-N446 hydrogen bonds form. Then, active-site base pairs dissociate one by one, and the RNA bases reanneal with the complementary base on the upstream DNA repeat. For each dissociating base pair a new one forms, thus conserving the number of base pairs during template translocation.

Alternative RNA degradation pathways by the exonuclease Pop2p from Saccharomyces cerevisiae.

The 3' to 5' exonuclease Pop2p (Caf1p) is part of the CCR4-NOT deadenylation complex that removes poly(A) tails from mRNAs in cells. Pop2p is structurally conserved in eukaryotes, but Saccharomyces cerevisiae Pop2p harbors noncanonical amino acids in its catalytic center. The enzymatic properties of S. cerevisiae Pop2p are not well defined. Here we characterize the RNA exonuclease activity of recombinant S. cerevisiae Pop2p. We find that S. cerevisiae Pop2p degrades RNAs via two alternative reactions pathways, one generating nucleotides with 5'-phosphates and RNA intermediates with 3'-hydroxyls, and the other generating nucleotides with 3'-phosphates and RNA intermediates with 3'-phosphates. The enzyme is not able to initiate the reaction on RNAs with a 3'-phosphate, which leads to accumulation of RNAs with 3'-phosphates that can exceed 10 nt and are resistant to further degradation by S. cerevisiae Pop2p. We further demonstrate that S. cerevisiae Pop2p degrades RNAs in three reaction phases: an initial distributive phase, a second processive phase and a third phase during which processivity gradually declines. We also show that mutations of subsets of amino acids in the catalytic center, including those previously thought to inactivate the enzyme, moderately reduce, but not eliminate activity. Only mutation of all five amino acids in the catalytic center diminishes activity of Pop2p to background levels. Collectively, our results reveal robust exonuclease activity of S. cerevisiae Pop2p with unusual enzymatic properties, characterized by alternative degradation pathways, multiple reaction phases and functional redundancy of amino acids in the catalytic core.

Epstein-Barr Virus Viral Processivity Factor EA-D Facilitates Virus Lytic Replication by Inducing Poly(ADP-Ribose) Polymerase 1 Degradation.

Gammaherpesviruses, including Epstein-Barr virus (EBV), are important human pathogens because they are associated with various tumors. Poly(ADP-ribose) polymerase 1 (PARP1) is a multifunctional host nuclear protein responsible for poly(ADP-ribosyl)ation (PARylation) of target proteins. While PARP1 acts as a negative regulator that suppresses the lytic replication of gammaherpesviruses, viruses are often equipped with various strategies to overcome PARP1 inhibition. However, the mechanisms of how EBV may modulate a repressive host protein, PARP1, are still elusive. In this study, we found that EBV reactivation induced PARP1 downregulation in EBV-infected cells. EBV DNA polymerase processivity factor EA-D, encoded by the BMRF1 gene, directly interacted with the central automodification domain (AD) of PARP1 and was necessary and sufficient to downregulate PARP1 via K29-linked polyubiquitination. Moreover, knockdown of EA-D in B95.8 cells restored PARP1 levels and abrogated the expression of ZTA (also known as ZEBRA), a switch molecule of the EBV life cycle during reactivation. Interestingly, PARP1 PARylated RTA, another key switch molecule, and decreased RTA transactivation on the promoters of the ZTA, BMRF1, and BMLF1 genes. EA-D alleviated the PARylation of RTA and further enhanced RTA-mediated transactivation of these lytic promoters in reporter assays. Taken together, our results suggest that EBV viral processivity factor plays a key role in facilitating lytic replication by inducing PARP1 degradation via its interaction with the PARP1 AD, which is a highly conserved mechanism among gammaherpesviruses to counteract host repressive activity of PARP1 against viral lytic replication. IMPORTANCE PARP1 acts as a negative regulator of lytic replication in EBV. To successfully enter the reactivation cycle, EBV has developed multiple strategies to counteract the host's repressive mechanisms. In this study, we investigated how EBV manipulated the host repressive factor PARP1 to facilitate lytic replication. The EBV processivity factor EA-D downregulated PARP1 in a proteasome-dependent manner via its direct binding with PARP1 AD. The knockdown of EA-D restored the PARP1 level and inhibited ZTA expression during reactivation. Interestingly, PARP1 PARylated RTA and EA-D reduced the PARylation of RTA, thereby promoting the ZTA promoter activity. These results suggest that EA-D plays a key role in EBV lytic replication by inducing PARP1 degradation in addition to supporting DNA replication as a viral processivity factor. Given that the KSHV processivity factor also induces PARP1 degradation and enhances RTA function, gammaherpesviruses share a conserved molecular mechanism to overcome the inhibitory effects of PARP1, promoting lytic replication.