scholarly journals DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Xuefeng Deng ◽  
Peng Xu ◽  
Wei Zou ◽  
Weiran Shen ◽  
Jianxin Peng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Respiratory Virus ◽  
Hek293 Cells ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Content Type ◽  
Dividing Cells ◽  
Cellular Dna ◽  
Replication Factors

ABSTRACT Human bocavirus 1 (HBoV1), an emerging human-pathogenic respiratory virus, is a member of the genus Bocaparvovirus of the Parvoviridae family. In human airway epithelium air-liquid interface (HAE-ALI) cultures, HBoV1 infection initiates a DNA damage response (DDR), activating all three phosphatidylinositol 3-kinase-related kinases (PI3KKs): ATM, ATR, and DNA-PKcs. In this context, activation of PI3KKs is a requirement for amplification of the HBoV1 genome (X. Deng, Z. Yan, F. Cheng, J. F. Engelhardt, and J. Qiu, PLoS Pathog, 12:e1005399, 2016, https://doi.org/10.1371/journal.ppat.1005399 ), and HBoV1 replicates only in terminally differentiated, nondividing cells. This report builds on the previous discovery that the replication of HBoV1 DNA can also occur in dividing HEK293 cells, demonstrating that such replication is likewise dependent on a DDR. Transfection of HEK293 cells with the duplex DNA genome of HBoV1 induces hallmarks of DDR, including phosphorylation of H2AX and RPA32, as well as activation of all three PI3KKs. The large viral nonstructural protein NS1 is sufficient to induce the DDR and the activation of the three PI3KKs. Pharmacological inhibition or knockdown of any one of the PI3KKs significantly decreases both the replication of HBoV1 DNA and the downstream production of progeny virions. The DDR induced by the HBoV1 NS1 protein does not cause obvious damage to cellular DNA or arrest of the cell cycle. Notably, key DNA replication factors and major DNA repair DNA polymerases (polymerase η [Pol η] and polymerase κ [Pol κ]) are recruited to the viral DNA replication centers and facilitate HBoV1 DNA replication. Our study provides the first evidence of the DDR-dependent parvovirus DNA replication that occurs in dividing cells and is independent of cell cycle arrest. IMPORTANCE The parvovirus human bocavirus 1 (HBoV1) is an emerging respiratory virus that causes lower respiratory tract infections in young children worldwide. HEK293 cells are the only dividing cells tested that fully support the replication of the duplex genome of this virus and allow the production of progeny virions. In this study, we demonstrate that HBoV1 induces a DDR that plays significant roles in the replication of the viral DNA and the production of progeny virions in HEK293 cells. We also show that both cellular DNA replication factors and DNA repair DNA polymerases colocalize within centers of viral DNA replication and that Pol η and Pol κ play an important role in HBoV1 DNA replication. Whereas the DDR that leads to the replication of the DNA of other parvoviruses is facilitated by the cell cycle, the DDR triggered by HBoV1 DNA replication or NS1 is not. HBoV1 is the first parvovirus whose NS1 has been shown to be able to activate all three PI3KKs (ATM, ATR, and DNA-PKcs).

scholarly journals Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Zekun Wang ◽  
Weiran Shen ◽  
Fang Cheng ◽  
Xuefeng Deng ◽  
John F. Engelhardt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Noncoding Rna ◽  
Rna Polymerase Iii ◽  
Human Bocavirus ◽  
Nonstructural Proteins ◽  
Viral Dna ◽  
Content Type ◽  
Small Noncoding Rna ◽  
Pol Iii ◽  
Tract Infections

ABSTRACT Human bocavirus 1 (HBoV1) belongs to the species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identified an additional HBoV1 gene, bocavirus-transcribed small noncoding RNA (BocaSR), within the 3′ noncoding region (nucleotides [nt] 5199 to 5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at levels similar to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we showed that BocaSR regulates the expression of HBoV1-encoded nonstructural proteins NS1, NS2, NS3, and NP1 but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for antiviral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors. IMPORTANCE Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identified a novel HBoV1 gene that lies in the 3′ noncoding region of the viral positive-sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nt. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing antiviral therapies.

Hairpin transfer-independent Parvovirus DNA Replication Produces Infectious Virus

2021 ◽  
Author(s):  
Weiran Shen ◽  
Zekun Wang ◽  
Kang Ning ◽  
Fang Cheng ◽  
John F. Engelhardt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Hek293 Cells ◽  
Progeny Virus ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Airway Epithelia ◽  
Viral Dna Replication ◽  
Human Airway ◽  
The Right ◽  
Human Airway Epithelia

Parvoviruses package a linear single-stranded DNA genome with hairpin structures at both ends. It has been thought that terminal hairpin sequences are indispensable for viral DNA replication. Here, we provide evidence that the hairpin-deleted duplex genomes of human bocavirus 1 (HBoV1) replicate in human embryonic kidney (HEK) 293 cells. We propose an alternative model for HBoV1 DNA replication in which the leading strand can initiate strand-displacement without “hairpin-transfer.” The transfection of the HBoV1 duplex genomes that retain a minimal replication origin at the right-end ( OriR ), but with extensive deletions in the right-end hairpin (REH), generated viruses in HEK293 cells at a level 10-20 times lower than the wild-type (WT) duplex genome. Importantly, these viruses that have a genome with various deletions after the OriR , but not the one retaining only the OriR , replicated in polarized human airway epithelia. We discovered that the 18-nt sequence (nt 5,403-5,420) beyond the OriR was sufficient to confer virus replication in polarized human airway epithelia, although its progeny virus production was ∼5 times lower than that of the WT virus. Thus, our study demonstrates that hairpin transfer-independent productive parvovirus DNA replication can occur. Importance Hairpin transfer-independent parvovirus replication was modeled with human bocavirus 1 (HBoV1) duplex genomes whose 5’ hairpin structure was ablated by various deletions. In HEK293 cells, these duplex viral genomes with ablated 5’/hairpin sequence replicated efficiently and generated viruses that productively infected polarized human airway epithelium. Thus, for the first time, we reveal a previously unknown phenomenon that the productive parvovirus DNA replication does not depend on the hairpin sequence at REH to initiate “rolling hairpin” DNA replication. Notably, the intermediates of viral DNA replication, as revealed two-dimensional electrophoresis, from transfections of hairpin sequence-deleted duplex genome and full-length genome in HEK293 cells, as well as from virus infection of polarized human airway epithelia are similar. Thus, the establishment of the hairpin transfer-independent parvoviral DNA replication deepens our understanding in viral DNA replication and may have implications in development of parvovirus-based viral vectors with alternative properties.

scholarly journals Human Parvovirus B19 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication

2013 ◽  
Vol 87 (23) ◽  
pp. 12766-12775 ◽  
Author(s):  
Yong Luo ◽  
Steve Kleiboeker ◽  
Xuefeng Deng ◽  
Jianming Qiu
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Content ◽  
Parvovirus B19 ◽  
S Phase ◽  
Human Parvovirus B19 ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cycle Arrest ◽  
Cellular Dna

Human parvovirus B19 (B19V) infection has a unique tropism to human erythroid progenitor cells (EPCs) in human bone marrow and the fetal liver. It has been reported that both B19V infection and expression of the large nonstructural protein NS1 arrested EPCs at a cell cycle status with a 4 N DNA content, which was previously claimed to be “G2/M arrest.” However, a B19V mutant infectious DNA (M20mTAD2) replicated well in B19V-semipermissive UT7/Epo-S1 cells but did not induce G2/M arrest (S. Lou, Y. Luo, F. Cheng, Q. Huang, W. Shen, S. Kleiboeker, J. F. Tisdale, Z. Liu, and J. Qiu, J. Virol.86:10748–10758, 2012). To further characterize cell cycle arrest during B19V infection of EPCs, we analyzed the cell cycle change using 5-bromo-2′-deoxyuridine (BrdU) pulse-labeling and DAPI (4′,6-diamidino-2-phenylindole) staining, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content. We found that although both B19V NS1 transduction and infection immediately arrested cells at a status of 4 N DNA content, B19V-infected 4 N cells still incorporated BrdU, indicating active DNA synthesis. Notably, the BrdU incorporation was caused neither by viral DNA replication nor by cellular DNA repair that could be initiated by B19V infection-induced cellular DNA damage. Moreover, several S phase regulators were abundantly expressed and colocalized within the B19V replication centers. More importantly, replication of the B19V wild-type infectious DNA, as well as the M20mTAD2mutant, arrested cells at S phase. Taken together, our results confirmed that B19V infection triggers late S phase arrest, which presumably provides cellular S phase factors for viral DNA replication.

scholarly journals Analysis ofcisandtransRequirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome

2016 ◽  
Vol 90 (17) ◽  
pp. 7761-7777 ◽  
Author(s):  
Weiran Shen ◽  
Xuefeng Deng ◽  
Wei Zou ◽  
John F. Engelhardt ◽  
Ziying Yan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Respiratory Tract Infections ◽  
Nonstructural Protein ◽  
Hek293 Cells ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
The Right ◽  
Tract Infections

ABSTRACTParvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriRin vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR.In vivostudies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication.IMPORTANCEHuman bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified bothcis-acting elements andtrans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin, which contains both NS1 nicking and binding sites, to a 46-nucleotide sequence in the right-end hairpin. The identification of these essential elements of HBoV1 DNA replication acting both incisand intranswill provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin and to design next-generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases.

scholarly journals Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Wei Zou ◽  
Zekun Wang ◽  
Min Xiong ◽  
Aaron Yun Chen ◽  
Peng Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Parvovirus B19 ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cycle Arrest ◽  
Damage Response ◽  
Cellular Dna

ABSTRACTHuman parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCEHuman parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.

scholarly journals Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Peter E. Burby ◽  
Lyle A. Simmons
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Genome Instability ◽  
Sos Response ◽  
Bacterial Cells ◽  
Cycle Progression ◽  
Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

scholarly journals Cellular Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Mediates Nuclear Import of Human Bocavirus 1 NP1 Protein and Modulates Viral Capsid Protein Expression

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Xiaomei Wang ◽  
Peng Xu ◽  
Fang Cheng ◽  
Yi Li ◽  
Zekun Wang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Capsid Protein ◽  
Nuclear Import ◽  
Nonstructural Protein ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Specificity Factor ◽  
Tract Infections

ABSTRACT Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication. IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development.

scholarly journals Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

2008 ◽  
Vol 82 (18) ◽  
pp. 9056-9064 ◽  
Author(s):  
Sally Roberts ◽  
Sarah R. Kingsbury ◽  
Kai Stoeber ◽  
Gillian L. Knight ◽  
Phillip H. Gallimore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Cellular Dna ◽  
In Cells

ABSTRACT Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

scholarly journals RB Reversibly Inhibits DNA Replication via Two Temporally Distinct Mechanisms

2004 ◽  
Vol 24 (12) ◽  
pp. 5404-5420 ◽  
Author(s):  
Steven P. Angus ◽  
Christopher N. Mayhew ◽  
David A. Solomon ◽  
Wesley A. Braden ◽  
Michael P. Markey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Cellular Proliferation ◽  
Negative Regulator ◽  
Proliferating Cells ◽  
Factor C ◽  
Replication Factors ◽  
Multiple Replication

ABSTRACT The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the “chronic” RB-mediated arrest state and the “acute” arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

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