The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

Background  

The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 ?.     

PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery.     

Diverse bacterial genomes encode an operon of two genes, one of which is an unusual class-I release factor that potentially recognizes atypical mRNA signals other than normal stop codons

Background  

While all codons that specify amino acids are universally recognized by tRNA molecules, codons signaling termination of translation are recognized by proteins known as class-I release factors (RF). In most eukaryotes and archaea a single RF accomplishes termination at all three stop codons. In most bacteria, there are two RFs with overlapping specificity, RF1 recognizes UA(A/G) and RF2 recognizes U(A/G)A.     

Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point<Superscript>1</Superscript>

Background  

Cytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing function of Mcm proteins, and a correlation between Mcm loading and cell cycle commitment for cells re-entering the cell cycle, led us to test the idea that cells expressing a defined high level of chromatin-bound Mcm6 in G1 are committed - i.e., past the G1 restriction point. We developed a cell-based assay for tightly-bound PCNA (PCNA*) and Mcm6 (Mcm6*), DNA content, and a mitotic marker to clearly define G1, S, G2, and M phases of the cell cycle. hTERT-BJ1, hTERT-RPE-1, and Molt4 cells were extracted with Triton X-100 followed by methanol fixation, stained with antibodies and DAPI, then measured by cytometry.     

An <Emphasis Type="Italic">in vivo</Emphasis> analysis of the localisation and interactions of human p66 DNA polymerase δ subunit

Background  

DNA polymerase δ is essential for eukaryotic DNA replication and also plays a role in DNA repair. The processivity of this polymerase complex is dependent upon its interaction with the sliding clamp PCNA and the polymerase-PCNA interaction is largely mediated through the p66 polymerase subunit. We have analysed the interactions of the human p66 DNA polymerase δ subunit with PCNA and with components of the DNA polymerase δ complex in vivo.     

Influence of HLA DRB1 alleles in the susceptibility of rheumatoid arthritis and the regulation of antibodies against citrullinated proteins and rheumatoid factor

Introduction  

The purpose of this study was to investigate the association between HLA-DRB1 alleles with susceptibility to rheumatoid arthritis (RA) and production of antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF).     

Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation

Background

PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome.

Scope

This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution.

Conclusions

Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.     

Analysis of the role of PCNA-DNA contacts during clamp loading

Background  

Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.     

Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast

Background  

Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner.     

Ancient diversification of eukaryotic MCM DNA replication proteins

Background  

Yeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7) for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear.     

Evolution of DNA Replication Protein Complexes in Eukaryotes and Archaea

Background

The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best.

Methodology/Principal Findings

While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex—all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea.

Conclusion/Significance

This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.     

Small molecule inhibitors of PCNA/PIP-box interaction suppress translesion DNA synthesis

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC₅₀ <5 μM inhibited cellular DNA replication at 10 μM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.     

Correlation between ribonucleoside-diphosphate reductase and three replication proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

There has long been evidence supporting the idea that RNR and the dNTP-synthesizing complex must be closely linked to the replication complex or replisome. We contributed to this body of evidence in proposing the hypothesis of the replication hyperstructure. A recently published work called this postulate into question, reporting that NrdB is evenly distributed throughout the cytoplasm. Consequently we were interested in the localization of RNR protein and its relationship with other replication proteins.     

Crystal Structure of the Shrimp Proliferating Cell Nuclear Antigen: Structural Complementarity with WSSV DNA Polymerase PIP-Box

DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV) that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA). This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein). The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.     

The microarchitecture of DNA replication domains

Most DNA synthesis in HeLa cell nucleus is concentrated in discrete foci. These synthetic sites can be identified by electron microscopy after allowing permeabilized cells to elongate nascent DNA in the presence of biotin-dUTP. Biotin incorporated into nascent DNA can be then immunolabeled with gold particles. Two types of DNA synthetic sites/replication factories can be distinguished at ultrastructural level: (1) electron-dense structures—replication bodies (RB), and (2) focal replication sites with no distinct underlying structure—replication foci (RF). The protein composition of these synthetic sites was studied using double immunogold labeling. We have found that both structures contain (a) proteins involved in DNA replication (DNA polymerase α, PCNA), (b) regulators of the cell cycle (cyclin A, cdk2), and (c) RNA processing components like Sm and SS-B/La auto antigens, p80-coilin, hnRNPs A1 and C1/C2. However, at least four regulatory and structural proteins (Cdk1, cyclin B1, PML and lamin B1) differ in their presence in RB and RF. Moreover, in contrast to RF, RB have structural organization. For example, while DNA polymerase α, PCNA and hnRNP A1 were diffusely spread throughout RB, hnRNP C1/C2 was found only at the very outside. Surprisingly, RB contained only small amounts of DNA. In conclusion, synthetic sites of both types contain similar but not the same sets of proteins. RB, however, have more developed microarchitecture, apparently with specific functional zones. This data suggest possible differences in genome regions replicated by these two types of replication factories.     

Mismatch Repair proteins are recruited to replicating DNA through interaction with Proliferating Cell Nuclear Antigen (PCNA)

Mismatch Repair (MMR) is closely linked to DNA replication; however, other than the role of the replicative sliding clamp (PCNA) in various MMR functions, the linkage between DNA replication and MMR has been difficult to investigate. Here we use an in vitro DNA replication system based on simian virus 40, to investigate MMR recruitment to replicating DNA. Both DNA replication and MMR proteins are recruited to replicating DNA in an origin-dependent fashion. Primer synthesis is required for recruitment of both PCNA and MMR proteins, but not for recruitment of the single-stranded DNA-binding protein (RPA). Blocking PCNA recruitment to replicating DNA with a p21-based polypeptide blocks PCNA and MMR, but not RPA recruitment. Once PCNA and subsequent proteins required for replication are loaded onto DNA, addition of p21 leaves PCNA on the replicating DNA, but actively displaces MMR proteins. These findings indicate that the MMR machinery is recruited to replicating DNA through its interaction with PCNA, and suggests that this occurs via binding of the MMR proteins to the multi-protein interaction sites on PCNA. These studies demonstrate the utility of this system for further investigation of the role of DNA replication in MMR.     

Baculovirus display of single chain antibody (scFv) using a novel signal peptide

Background  

Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.     

Involvement of TIP60 acetyltransferase in intracellular <Emphasis Type="Italic">Salmonella</Emphasis>replication

Background  

Salmonella enterica is a facultative intracellular pathogen that replicates within a membrane-bound compartment termed Salmonella containing vacuole (SCV). The biogenesis of SCV requires Salmonella type III protein secretion/translocation system and their effector proteins which are translocated into host cells to exploit the vesicle trafficking pathways. SseF is one of these effectors required for SCV formation and Intracellular Salmonella replication through unknown mechanisms.