scholarly journals Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins

2007 ◽  
Vol 82 (3) ◽  
pp. 1458-1464 ◽  
Author(s):  
Kevin Nash ◽  
Weijun Chen ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Productive Infection ◽  
Nuclear Antigen ◽  
Minichromosome Maintenance ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Mcm Complex ◽  
Factor C

ABSTRACT Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase δ as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase δ, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase δ inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase δ play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.

scholarly journals Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein

2008 ◽  
Vol 83 (1) ◽  
pp. 454-469 ◽  
Author(s):  
Kevin Nash ◽  
Weijun Chen ◽  
Max Salganik ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Protein Complexes ◽  
Protein Translation ◽  
Cellular Protein ◽  
Productive Infection ◽  
Cellular Proteins ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Rep Proteins

ABSTRACT Adeno-associated virus (AAV) codes for four related nonstructural Rep proteins. AAV both replicates and assembles in the nucleus and requires coinfection with a helper virus, either adenovirus (Ad) or herpesvirus, for a productive infection. Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins, which are known to be directly involved in DNA replication, control of viral and cellular transcription, splicing, and protein translation. In this study, we used affinity-tagged Rep protein to purify cellular protein complexes that were associated with Rep in cells that had been infected with Ad and AAV. In all, we identified 188 cellular proteins from 16 functional categories, including 14 transcription factors, 6 translation factors, 15 potential splicing proteins, 5 proteins involved in protein degradation, and 13 proteins involved in DNA replication or repair. This dramatically increases the number of potential interactions over the current number of approximately 26. Twelve of the novel proteins found were further tested by coimmunoprecipitation or colocalization using confocal immunomicroscopy. Of these, 10 were confirmed as proteins that formed complexes with Rep, including proteins of the MCM complex (DNA replication), RCN1 (membrane transport), SMC2 (chromatin dynamics), EDD1 (ubiquitin ligase), IRS4 (signal transduction), and FUS (splicing). Computer analysis suggested that 45 and 28 of the 188 proteins could be placed in a pathway of interacting proteins involved in DNA replication and protein synthesis, respectively. Of the proteins involved in DNA replication, all of the previously identified proteins involved in AAV DNA replication were found, except Ad DBP. The only Ad protein found to interact with Rep was the E1b55K protein. In addition, we confirmed that Rep interacts with Ku70/80 helicase. In vitro DNA synthesis assays demonstrated that although Ku helicase activity could substitute for MCM to promote strand displacement synthesis, its presence was not essential. Our study suggests that the interaction of AAV with cellular proteins is much more complex than previously suspected and provides a resource for further studies of the AAV life cycle.

scholarly journals Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection

1998 ◽  
Vol 72 (4) ◽  
pp. 2777-2787 ◽  
Author(s):  
Tie-Hua Ni ◽  
William F. McDonald ◽  
Irene Zolotukhin ◽  
Thomas Melendy ◽  
Shou Waga ◽  
...  
Keyword(s):  
Dna Replication ◽  
Hela Cell ◽  
Dna Polymerase ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Cell Extracts ◽  
Terminal Repeats ◽  
Factor C

ABSTRACT We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and ɛ, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase ɛ for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

scholarly journals Visualization of replication initiation and elongation in Drosophila

2002 ◽  
Vol 159 (2) ◽  
pp. 225-236 ◽  
Author(s):  
Julie M. Claycomb ◽  
David M. MacAlpine ◽  
James G. Evans ◽  
Stephen P. Bell ◽  
Terry L. Orr-Weaver
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Developmental Stages ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Minichromosome Maintenance ◽  
Egg Chamber ◽  
Two Phases ◽  
Replication Factors

Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2–7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre–replication complex component, double-parked protein/cell division cycle 10–dependent transcript 1, is not only necessary for proper MCM2–7 localization, but, unexpectedly, is present during elongation.

scholarly journals Regulation of RelA (p65) Function by the Large Subunit of Replication Factor C

2003 ◽  
Vol 23 (2) ◽  
pp. 721-732 ◽  
Author(s):  
Lisa A. Anderson ◽  
Neil D. Perkins
Keyword(s):  
Tumor Suppressor ◽  
Large Subunit ◽  
Dominant Negative ◽  
Nuclear Antigen ◽  
Replication Factor C ◽  
Clamp Loader ◽  
Replication Factor ◽  
Factor C

ABSTRACT The RelA (p65) subunit of NF-κB is an important regulator of inflammation, proliferation, and apoptosis. We have discovered that the large subunit, p140, of replication factor C (RFC) can function as a regulator of RelA. RFC is a clamp loader, facilitating the addition and removal of proliferating-cell nuclear antigen from DNA during replication and repair but can also interact directly with the retinoblastoma tumor suppressor protein and the transcription factor C/EBPα. We find that RFC (p140) interacts with RelA both in vitro and in vivo and stimulates RelA transactivation. In contrast, coexpression of fragments of RFC (p140) that mediate the interaction with RelA results in transcriptional inhibition. The significance of this regulation was confirmed by using short interfering RNA oligonucleotides targeted to RFC (p140). Down regulation of endogenous RFC (p140) inhibits expression from a chromosomally integrated reporter plasmid induced by endogenous, TNF-α-activated NF-κB. Dominant negative fragments of RFC (p140) also cooperate with overexpressed RelA to induce cell death. Interestingly, RFC (p140) also interacts with the tumor suppressor p53. Taken together, these observations suggest that, in addition to its previously described function in DNA replication and repair, RFC (p140) has an important role as a regulator of transcription and NF-κB activity.

scholarly journals Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

2005 ◽  
Vol 79 (1) ◽  
pp. 28-38 ◽  
Author(s):  
John M. Casper ◽  
Jennifer M. Timpe ◽  
John David Dignam ◽  
James P. Trempe
Keyword(s):  
Gene Expression ◽  
Random Sequence ◽  
Gene Promoter ◽  
Helper Virus ◽  
Host Cells ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

scholarly journals The RAD52 Recombinational Repair Pathway is Essential in pol30 (PCNA) Mutants That Accumulate Small Single-Stranded DNA Fragments During DNA Synthesis

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 611-624 ◽  
Author(s):  
Bradley J Merrill ◽  
Connie Holm
Keyword(s):  
Dna Replication ◽  
Synthetic Lethality ◽  
Large Subunit ◽  
Group Analysis ◽  
Nuclear Antigen ◽  
Recombinational Repair ◽  
Dna Fragments ◽  
Single Stranded Dna ◽  
Factor C

Abstract To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation. We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52, and rad57). We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1Δ) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.

scholarly journals Analysis of mutations in adeno-associated virus Rep protein in vivo and in vitro.

1992 ◽  
Vol 66 (7) ◽  
pp. 4050-4057 ◽  
Author(s):  
D M McCarty ◽  
T H Ni ◽  
N Muzyczka
Keyword(s):  
Adeno Associated Virus ◽  
Rep Protein

scholarly journals The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome

2015 ◽  
Vol 290 (34) ◽  
pp. 20919-20933 ◽  
Author(s):  
Pavana M. Hegde ◽  
Arijit Dutta ◽  
Shiladitya Sengupta ◽  
Joy Mitra ◽  
Sanjay Adhikari ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Dna Glycosylase ◽  
Nuclear Antigen ◽  
Replication Factor C ◽  
Replication Factor ◽  
Terminal Domain ◽  
Human Dna ◽  
Factor C

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.

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