Nascent DNA Research Articles

Checkpoint phosphorylation sites on budding yeast Rif1 protect nascent DNA from degradation by Sgs1-Dna2.

In budding yeast the Rif1 protein is important for protecting nascent DNA at blocked replication forks, but the mechanism has been unclear. Here we show that budding yeast Rif1 must interact with Protein Phosphatase 1 to protect nascent DNA. In the absence of Rif1, removal of either Dna2 or Sgs1 prevents nascent DNA degradation, implying that Rif1 protects nascent DNA by targeting Protein Phosphatase 1 to oppose degradation by the Sgs1-Dna2 nuclease-helicase complex. This functional role for Rif1 is conserved from yeast to human cells. Yeast Rif1 was previously identified as a target of phosphorylation by the Tel1/Mec1 checkpoint kinases, but the importance of this phosphorylation has been unclear. We find that nascent DNA protection depends on a cluster of Tel1/Mec1 consensus phosphorylation sites in the Rif1 protein sequence, indicating that the intra-S phase checkpoint acts to protect nascent DNA through Rif1 phosphorylation. Our observations uncover the pathway by which budding yeast Rif1 stabilises newly synthesised DNA, highlighting the crucial role Rif1 plays in maintaining genome stability from lower eukaryotes to humans.

DCAF14 regulates CDT2 to promote SET8-dependent replication fork protection.

DDB1- and CUL4-associated factors (DCAFs) CDT2 and DCAF14 are substrate receptors for Cullin4-RING E3 ubiquitin ligase (CRL4) complexes. CDT2 is responsible for PCNA-coupled proteolysis of substrates CDT1, p21, and SET8 during S-phase of cell cycle. DCAF14 functions at stalled replication forks to promote genome stability, but the mechanism is unknown. We find that DCAF14 mediates replication fork protection by regulating CRL4CDT2 activity. Absence of DCAF14 causes increased proteasomal degradation of CDT2 substrates. When forks are challenged with replication stress, increased CDT2 function causes stalled fork collapse and impairs fork recovery in DCAF14-deficient conditions. We further show that stalled fork protection is dependent on CDT2 substrate SET8 and does not involve p21 and CDT1. Like DCAF14, SET8 blocks nuclease-mediated digestion of nascent DNA at remodeled replication forks. Thus, unregulated CDT2-mediated turnover of SET8 triggers nascent strand degradation when DCAF14 is absent. We propose that DCAF14 controls CDT2 activity at stalled replication forks to facilitate SET8 function in safeguarding genomic integrity.

YAP1-Dependent Promotion of Downstream Transcription through BRD4 Mediates Selinexor Resistance in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent subtype of non-Hodgkin lymphoma. Despite advancements in treatment, the prognosis for relapsed and/or refractory DLBCL (R/R DLBCL) patients remains unfavorable due to the development of drug resistance and disease progression. The pressing need to enhance the outcomes in R/R DLBCL patients necessitates ongoing investigations into novel therapeutic approaches. Among various exportins, the nuclear export protein exportin 1 (XPO1) solely governs the transportation of nuclear proteins harboring leucine-rich hydrophobic nuclear export signals (NES), including essential regulators like p53, RB1, and p27. Selinexor, an oral inhibitor specifically targeting XPO1, has emerged as a promising candidate in R/R DLBCL patients. Encouraging findings from a phase II clinical trial demonstrate a more favorable efficacy of Selinexor in the germinal center B-cell (GCB) subtype compared with that in other subtypes of DLBCL. Nonetheless, the clinical implementation of Selinexor has led to the emergence of acquired resistance, posing a significant challenge for medical practitioners. Efforts to overcome this resistance and establish strategies to maximize Selinexor's therapeutic potential remain of paramount importance in addressing the urgent clinical needs of R/R DLBCL patients. Methods: Characterization of Selinexor resistance targets was performed using a CRISPR/Cas9 screen transcription factors library pool at 0-day and 14-day time points after treatment. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. Synergistic treatment approach utilizing BRD4 inhibitor JQ1, known for its curative effects in AML, MM, and ALL, in combination with Selinexor were also evaluated. Mechanistic exploration of BRD4's role in Selinexor resistance was further analyzed via RNA-seq, ChIP-seq, as well as luciferase reporter experiments. Therapeutic outcomes were evaluated in R/R DLBCL patients undergoing Selinexor treatment via PET/CT imaging. Results ： BRD4, BRD7, ZMYND8, and YTHDC1 were identified as top Selinexor resistance candidate targets through CRISPR/Cas9 Screen. The knock-down of BRD4 enhanced the killing ability of Selinexor in vitro and reduced tumor burden in subcutaneously transplanted tumor mice after treatment with Selinexor in vivo in DLBCL. The combined treatment with BRD4 inhibitor JQ1 and Selinexor had a stronger ability to promote cell apoptosis, enhance DNA damage and reduce nascent DNA compared with the single drug group. Hematological and organ toxicity tests confirmed an acceptable toxicity for the combined drug regimen. ChIP-seq differential peaks and RNA-seq differential genes analysis elucidated the crucial role of YAP1's sub-cellular localization under Selinexor treatment. YAP1 nuclear translocation enhanced transcription and formed an interaction with BRD4, resulting in augmented BRD4 binding domain, and finally lead to the resistance of Selinexor. PET/CT imaging showed that one patient with high XPO1 expression achieved complete response (CR), exhibiting a sensitivity to Selinexor treatment, but the other exhibits resistance associated with a lack of XPO1 expression and BRD4-regulated acetylation. Conclusions: The nuclear translocation of YAP1 and its interaction with BRD4 mediated the transcriptional regulation and further induced resistance to Selinexor in DLBCL. BRD4 inhibitor JQ1 combined with low-dose Selinexor can mitigate blood toxicity and enhance therapeutic efficacy, achieving an improved outcome.

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.

Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart.

R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.

Modified iPOND revealed the role of mutant p53 in promoting helicase function and telomere maintenance.

The G-rich DNA, such as telomere, tends to form G-quadruplex (G4) structure, which slows down the replication fork progression, induces replication stress, and becomes the chromosome fragile sites. Here we described a molecular strategy that cells developed to overcome the DNA replication stress via DNA helicase regulation. The p53N236S (p53S) mutation has been found in the Werner syndrome mouse embryo fibroblast (MEFs) escaped from senescence, could be the driving force for cell escaping senescence. We revealed that the p53S could transcriptionally up-regulate DNA helicases expression, including Wrn, Blm, Timeless, Ddx, Mcm, Gins, Fanc, as well as telomere specific proteins Terf1, Pot1, through which p53S promoted the unwinding of G4 structures, and protected the cells from DNA replication stress induced by G4 stabilizer. By modified iPOND (isolation of proteins on nascent DNA) assay and telomere assay, we demonstrated that the p53S could promote the recruitment of those helicases to the DNA replication forks, facilitated the maintenance of telomere, and prevent the telomere dysfunction induced by G4 stabilizer. Interestingly, we did not observe the function of promoting G4 resolving and facilitating telomere lengthening in the cells with Li-Fraumeni Syndrome mutation-p53R172H (p53H), which suggests that this is the specific gain of function for p53S. Together our data suggest that the p53S could gain the new function of releasing the replication stress via regulating the helicase function and G4 structure, which benefits telomere lengthening. This strategy could be applied to the treatment of diseases caused by telomere replication stress.

The cancer testes antigen, HORMAD1, limits genomic instability in cancer cells by protecting stalled replication forks

Tumors anomalously induce the expression of meiotic genes, which are otherwise restricted only to developing gametes. If and how these aberrantly expressed meiotic proteins influence DNA metabolism is not clear, but could have important implications for how tumors acquire and mitigate genomic instability. HORMAD1 is a highly conserved meiotic protein that is frequently expressed in lung adenocarincoma where its expression correlates with reduced patient survival and increased mutation burden. Here, we find that HORMAD1 associates with the replisome and is critical for protecting stalled DNA replication forks. Loss of HORMAD1 leads to nascent DNA strand degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. We find that these phenotypes are due to limited RAD51 loading onto stalled replication forks in the absence of HORMAD1. Ultimately, loss of HORMAD1 leads to increased DNA breaks and chromosomal defects, which is exacerbated dramatically by induction of replication stress. Tumor cells proliferate despite encountering chronic replication stress, placing them on the precipice of catastrophic genomic damage. Our data support the hypothesis that the aberrant expression of HORMAD1 is engaged to attenuate the accumulation of excessive DNA damage due to chronic replication stress, which may otherwise lead to accumulation of toxic levels of genomic instability.

Epigenetic modifications evidenced by isolation of proteins on nascent DNA and immunofluorescence in hydroxyurea-treated root meristem cells of Vicia faba

Main conclusionBy implementation of the iPOND technique for plant material, changes in posttranslational modifications of histones were identified in hydroxyurea-treated root meristem cells of Vicia. Replication stress (RS) disrupts or inhibits replication forks and by altering epigenetic information of the newly formed chromatin can affect gene regulation and/or spatial organisation of DNA. Experiments on Vicia faba root meristem cells exposed to short-term treatment with 3 mM hydroxyurea (HU, an inhibitor of DNA replication) were aimed to understand epigenetic changes related to RS. To achieve this, the following histone modifications were studied using isolation of proteins on nascent DNA (iPOND) technique (for the first time on plant material) combined with immunofluorescence labeling: (i) acetylation of histone H3 at lysine 56 (H3K56Ac), (ii) acetylation of histone H4 at Lys 5 (H4K5Ac), and (iii) phosphorylation of histone H3 at threonine 45 (H3T45Ph). Certainly, the implementation of the iPOND method for plants may prove to be a key step for a more in-depth understanding of the cell's response to RS at the chromatin level.

Persistent Acetylation of Histone H3 Lysine 56 Compromises the Activity of DNA Replication Origins.

In Saccharomyces cerevisiae, newly synthesized histones H3 are acetylated on lysine 56 (H3 K56ac) by the Rtt109 acetyltransferase prior to their deposition on nascent DNA behind replication forks. Two deacetylases of the sirtuin family, Hst3 and Hst4, remove H3 K56ac from chromatin after S phase. hst3Δ hst4Δ cells present constitutive H3 K56ac, which sensitizes cells to replicative stress via unclear mechanisms. A chemogenomic screen revealed that DBF4 heterozygosity sensitizes cells to NAM-induced inhibition of sirtuins. DBF4 and CDC7 encode subunits of the Dbf4-dependent kinase (DDK), which activates origins of DNA replication during S phase. We show that (i) cells harboring the dbf4-1 or cdc7-4 hypomorphic alleles are sensitized to NAM, and that (ii) the sirtuins Sir2, Hst1, Hst3, and Hst4 promote DNA replication in cdc7-4 cells. We further demonstrate that Rif1, an inhibitor of DDK-dependent activation of origins, causes DNA damage and replication defects in NAM-treated cells and hst3Δ hst4Δ mutants. cdc7-4 hst3Δ hst4Δ cells are shown to display delayed initiation of DNA replication, which is not due to intra-S checkpoint activation but requires Rtt109-dependent H3 K56ac. Our results suggest that constitutive H3 K56ac sensitizes cells to replicative stress in part by negatively influencing the activation of origins of DNA replication.

Multi-step processing of replication stress-derived nascent strand DNA gaps by MRE11 and EXO1 nucleases

Accumulation of single stranded DNA (ssDNA) gaps in the nascent strand during DNA replication has been associated with cytotoxicity and hypersensitivity to genotoxic stress, particularly upon inactivation of the BRCA tumor suppressor pathway. However, how ssDNA gaps contribute to genotoxicity is not well understood. Here, we describe a multi-step nucleolytic processing of replication stress-induced ssDNA gaps which converts them into cytotoxic double stranded DNA breaks (DSBs). We show that ssDNA gaps are extended bidirectionally by MRE11 in the 3’−5’ direction and by EXO1 in the 5’−3’ direction, in a process which is suppressed by the BRCA pathway. Subsequently, the parental strand at the ssDNA gap is cleaved by the MRE11 endonuclease generating a double strand break. We also show that exposure to bisphenol A (BPA) and diethylhexyl phthalate (DEHP), which are widespread environmental contaminants due to their use in plastics manufacturing, causes nascent strand ssDNA gaps during replication. These gaps are processed through the same mechanism described above to generate DSBs. Our work sheds light on both the relevance of ssDNA gaps as major determinants of genomic instability, as well as the mechanism through which they are processed to generate genomic instability and cytotoxicity.

Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress.

Interferon restores replication fork stability and cell viability in BRCA-defective cells via ISG15

DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that basal levels of the IFN-stimulated gene 15, ISG15, and its conjugation (ISGylation) are essential to protect nascent DNA from degradation. Moreover, IFNβ treatment restores replication fork stability in BRCA1/2-deficient cells, which strictly depends on topoisomerase-1, and rescues lethality of BRCA2-deficient mouse embryonic stem cells. Although IFNβ activates hundreds of genes, these effects are specifically mediated by ISG15 and ISGylation, as their inactivation suppresses the impact of IFNβ on DNA replication. ISG15 depletion significantly reduces cell proliferation rates in human BRCA1-mutated triple-negative, whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, which we propose as an in-built mechanism of drug resistance linked to BRCAness.

ATR inhibition induces synthetic lethality in mismatch repair-deficient cells and augments immunotherapy.

The mismatch repair (MMR) deficiency of cancer cells drives mutagenesis and offers a useful biomarker for immunotherapy. However, many MMR-deficient (MMR-d) tumors do not respond to immunotherapy, highlighting the need for alternative approaches to target MMR-d cancer cells. Here, we show that inhibition of the ATR kinase preferentially kills MMR-d cancer cells. Mechanistically, ATR inhibitor (ATRi) imposes synthetic lethality on MMR-d cells by inducing DNA damage in a replication- and MUS81 nuclease-dependent manner. The DNA damage induced by ATRi is colocalized with both MSH2 and PCNA, suggesting that it arises from DNA structures recognized by MMR proteins during replication. In syngeneic mouse models, ATRi effectively reduces the growth of MMR-d tumors. Interestingly, the antitumor effects of ATRi are partially due to CD8+ T cells. In MMR-d cells, ATRi stimulates the accumulation of nascent DNA fragments in the cytoplasm, activating the cGAS-mediated interferon response. The combination of ATRi and anti-PD-1 antibody reduces the growth of MMR-d tumors more efficiently than ATRi or anti-PD-1 alone, showing the ability of ATRi to augment the immunotherapy of MMR-d tumors. Thus, ATRi selectively targets MMR-d tumor cells by inducing synthetic lethality and enhancing antitumor immunity, providing a promising strategy to complement and augment MMR deficiency-guided immunotherapy.

Transcription and replication: walking a genomic tightrope hand-in-hand

A recent study by Fenstermaker et al. in Nature describes how transcriptionally active RNA polymerase II (Pol II) clings to the genomic tightrope during the passage of the replication fork and rapidly resumes transcription of immature RNA from both strands of nascent DNA, facilitated by protein–protein interactions between the replication and transcription machineries.

Hot on RAD51C: structure and functions of RAD51C-XRCC3.

A new study by Longo, Roy et al. has solved the structure of the RAD51C-XRCC3 (CX3) heterodimer with a bound ATP analog, identifying two main structural interfaces and defining separable replication fork stability roles. One function relates to the ability of RAD51C to bind and assemble CX3 on nascent DNA, with impact on the ability of forks to restart upon replication stress. The other relates to effective CX3 heterodimer formation, required for 5' RAD51 filament capping, with effects on RAD51 filament disassembly, fork protection and influencing the persistence of reversed forks.

The COMPASS subunit Spp1 protects nascent DNA at the Tus/Ter replication fork barrier by limiting DNA availability to nucleases

Homologous recombination factors play a crucial role in protecting nascent DNA during DNA replication, but the role of chromatin in this process is largely unknown. Here, we used the bacterial Tus/Ter barrier known to induce a site-specific replication fork stalling in S. cerevisiae. We report that the Set1C subunit Spp1 is recruited behind the stalled replication fork independently of its interaction with Set1. Spp1 chromatin recruitment depends on the interaction of its PHD domain with H3K4me3 parental histones deposited behind the stalled fork. Its recruitment prevents the accumulation of ssDNA at the stalled fork by restricting the access of Exo1. We further show that deleting SPP1 increases the mutation rate upstream of the barrier favoring the accumulation of microdeletions. Finally, we report that Spp1 protects nascent DNA at the Tus/Ter stalled replication fork. We propose that Spp1 limits the remodeling of the fork, which ultimately limits nascent DNA availability to nucleases.

Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides.

A modified 2'-deoxycytidine triphosphate derivative (dCTOTP) bearing thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was found to incorporate into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dCTOTP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dCTOTP. When dCTOTP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor in real time the cells that were actively synthesizing DNA from the very first moments after treatment. We anticipate that this probe can be used to study chromatin organization and dynamics.

Real‐time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA‐Incorporated Thiazole Orange‐Modified Nucleotides

AbstractA modified 2′‐deoxycytidine triphosphate derivative (dCTOTP) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dCTOTP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non‐incorporated (i.e. free and non‐covalently intercalated) forms of dCTOTP. When dCTOTP was delivered into live U‐2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.

Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection.

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes.