Ellen Fanning

A dynamic model for replication protein A (RPA) function in DNA processing pathways

Processing of DNA in replication, repair and recombination pathways in cells of all organisms requires the participation of at least one major single-stranded DNA (ssDNA)-binding protein. This protein protects ssDNA from nucleolytic damage, prevents hairpin formation and blocks DNA reannealing until the processing pathway is successfully completed. Many ssDNA-binding proteins interact physically and functionally with a variety of other DNA processing proteins. These interactions are thought to temporally order and guide the parade of proteins that ‘trade places’ on the ssDNA, a model known as ‘hand-off’, as the processing pathway progresses. How this hand-off mechanism works remains poorly understood. Recent studies of the conserved eukaryotic ssDNA-binding protein replication protein A (RPA) suggest a novel mechanism by which proteins may trade places on ssDNA by binding to RPA and mediating conformation changes that alter the ssDNA-binding properties of RPA. This article reviews the structure and function of RPA, summarizes recent studies of RPA in DNA replication and other DNA processing pathways, and proposes a general model for the role of RPA in protein-mediated hand-off.

Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen.

The purified human single‐stranded DNA binding protein, replication protein A (RP‐A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha‐primase, as shown by ELISA and a modified immunoblotting technique. RP‐A associated efficiently with the isolated primase, as well as with intact polymerase alpha‐primase. The 70 kDa subunit of RP‐A was sufficient for association with polymerase alpha‐primase. Purified SV40 large T antigen bound to intact RP‐A and to polymerase‐primase, but not to any of the separated subunits of RP‐A or to the isolated primase. These results suggest that the specific protein‐protein interactions between RP‐A, polymerase‐primase and T antigen may play a role in the initiating of SV40 DNA replication.

Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen

The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting α-helices to expand/constrict the channel, producing an ‘iris’ effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.

SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.

Purified SV40 large T antigen and purified DNA polymerase alpha‐primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase‐T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10‐fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate‐specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein‐protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.

RPA2 Is a Direct Downstream Target for ATR to Regulate the S-phase Checkpoint

Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.

Ataxia Telangiectasia-Mutated Damage-Signaling Kinase- and Proteasome-Dependent Destruction of Mre11-Rad50-Nbs1 Subunits in Simian Virus 40-Infected Primate Cells

ABSTRACT Although the mechanism of simian virus 40 (SV40) DNA replication has been extensively investigated with cell extracts, viral DNA replication in productively infected cells utilizes additional viral and host functions whose interplay remains poorly understood. We show here that in SV40-infected primate cells, the activated ataxia telangiectasia-mutated (ATM) damage-signaling kinase, γ-H2AX, and Mre11-Rad50-Nbs1 (MRN) assemble with T antigen and other viral DNA replication proteins in large nuclear foci. During infection, steady-state levels of MRN subunits decline, although the corresponding mRNA levels remain unchanged. A proteasome inhibitor stabilizes the MRN complex, suggesting that MRN may undergo proteasome-dependent degradation. Analysis of mutant T antigens with disrupted binding to the ubiquitin ligase CUL7 revealed that MRN subunits are stable in cells infected with mutant virus or transfected with mutant viral DNA, implicating CUL7 association with T antigen in MRN proteolysis. The mutant genomes produce fewer virus progeny than the wild type, suggesting that T antigen-CUL7-directed proteolysis facilitates virus propagation. Use of a specific ATM kinase inhibitor showed that ATM kinase signaling is a prerequisite for proteasome-dependent degradation of MRN subunits as well as for the localization of T antigen and damage-signaling proteins to viral replication foci and optimal viral DNA replication. Taken together, the results indicate that SV40 infection manipulates host DNA damage-signaling to reprogram the cell for viral replication, perhaps through mechanisms related to host recovery from DNA damage.

An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase.

DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase α. Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase are lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified, and characterized. CD and NMR spectroscopy experiments demonstrated that p58C forms a well folded structure. The protein has a distinctive brownish color, and evidence from inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy revealed characteristics consistent with the presence of a [4Fe-4S] high potential iron protein cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wild-type protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first suggestion that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA-processing machinery.

The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

ABSTRACT To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-β) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-β in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-β region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-β deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-β region is sufficient to direct initiation and that specific DNA sequences in the ori-β region are required for efficient initiation activity.

Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation, and PCNA itself tethers DNA polymerase delta to DNA.

Replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA) assemble a complex, called sliding clamp, onto DNA. The clamp in turn loads DNA polymerases (pol) δ and ε to form the corresponding holoenzymes, which play an essential role in replication of eukaryotic chromosomal DNA and in several DNA repair pathways. To determine the fate of RF-C after loading of PCNA onto DNA, we tagged the RF-C subunit p37 with a protein kinase A recognition motif, so that the recombinant five-subunit RF-C complex could be32P-labeled and quantitatively detected in femtomolar amounts. Nonspecific binding of RF-C to DNA was minimized by replacing the p140 subunit with an N-terminally truncated p140 subunit lacking the previously identified nonspecific DNA binding domain. Neither of these modifications impaired the clamp loading activity of the recombinant RF-C. Using gel filtration techniques, we demonstrated that RF-C dissociated from the DNA after clamp loading or pol δ holoenzyme assembly, while PCNA or PCNA·pol δ complex remained bound to DNA. PCNA catalytically loaded onto the template-primer was sufficient by itself to tether pol δ and stimulate DNA replication. The readdition of RF-C to the isolated PCNA·DNA complex did not further stimulate pol δ DNA synthesis. We conclude that pol δ holoenzyme consists of PCNA and pol δ core and that RF-C serves only to load PCNA clamp.

The replicon revisited: an old model learns new tricks in metazoan chromosomes

The origins of DNA replication were proposed in the replicon model to be specified genetically by replicator elements that coordinate the initiation of DNA synthesis with gene expression and cell growth. Recent studies have identified DNA sequences in mammalian cells that fulfil the genetic criteria for replicators and are beginning to uncover the sequence requirements for the initiation of DNA replication. Mammalian replicators are composed of non‐redundant modules that cooperate to direct initiation to specific chromosomal sites. Conversely, replicators do not show strong sequence similarity, and their ability to initiate replication depends on the chromosomal context and epigenetic factors, as well as their primary sequence. Here, we review the properties of metazoan replicators, and discuss the genetic and epigenetic factors that determine where and when DNA replication is initiated.