RNA‐Directed rna polymerases of plants

The report in 1971 by Comuet and Astier‐Manifacier that Chinese cabbage contains an active RNA‐dependent RNA polymerase has been extended to all plants studied. This has met with much opposition because the central dogma of molecular biology requires no replication mechanism for RNA. Only upon RNA virus infection are such enzymes needed, and it was generally believed that these were always and only virus‐coded. The purification and characterization of several of these plant viruses will be reviewed, with particular reference to the fact that while their amount in plant tissue is variably increased by various RNA virus infections their nature is unaffected by the viral genome and is strictly host‐specific. It will be noted, however, that in a specific instance viral infection has been shown to affect an important property of the enzyme. Also, it has become evident that certain plant viruses resemble animal picorna viruses (e.g., polio virus) and that these viruses carry an RNA polymerase gene. The same may be true, but has not been proven, for a small group of plant viruses that shows resemblances to the prokaryotic RNA phages in which a viral gene product together with host proteins form the RNA polymerase. An important question that remains to be solved in future work is the role of RNA polymerases in normal plant cell biology. Also, the mechanism by which viral infection causes the enzyme to become largely membrane or organelle bound and possibly conformationally changed in the process remains to be elucidated.     

Differential inactivation of DNA polymerases α and β by aldehyde compounds

The effects of cyclohexanecarboxaldehyde, benzaldehyde and protocatechualdehyde on the activities of DNA polymerases α, β and E. coli DNA polymerase I were investigated. On direct addition of the aldehydes to the DNA polymerase assay mixture containing activated DNA or poly(dA) (dT)_12–18 as a template, DNA polymerase α was most strongly inhibited by the aldehyde compounds, while DNA polymerases β and I were resistant to such aldehyde inhibition. On preincubation of the enzymes with aldehyde, both DNA polymerases α and β were inactivated; however, DNA polymerase β was protected from the inactivation when activated DNA was added to the preincubation mixture. The inhibition of DNA polymerase α by aldehyde was noncompetitive with regard to the substrate dNTP and competitive with regard to the template DNA. The extent of inhibition of DNA polymerase α by aldehyde was partly reduced by the addition of cysteine to the reaction mixture.     

Translesion DNA polymerases in eukaryotes: what makes them tick?

Life as we know it, simply would not exist without DNA replication. All living organisms utilize a complex machinery to duplicate their genomes and the central role in this machinery belongs to replicative DNA polymerases, enzymes that are specifically designed to copy DNA. “Hassle-free” DNA duplication exists only in an ideal world, while in real life, it is constantly threatened by a myriad of diverse challenges. Among the most pressing obstacles that replicative polymerases often cannot overcome by themselves are lesions that distort the structure of DNA. Despite elaborate systems that cells utilize to cleanse their genomes of damaged DNA, repair is often incomplete. The persistence of DNA lesions obstructing the cellular replicases can have deleterious consequences. One of the mechanisms allowing cells to complete replication is “Translesion DNA Synthesis (TLS)”. TLS is intrinsically error-prone, but apparently, the potential downside of increased mutagenesis is a healthier outcome for the cell than incomplete replication. Although most of the currently identified eukaryotic DNA polymerases have been implicated in TLS, the best characterized are those belonging to the “Y-family” of DNA polymerases (pols η, ι, κ and Rev1), which are thought to play major roles in the TLS of persisting DNA lesions in coordination with the B-family polymerase, pol ζ. In this review, we summarize the unique features of these DNA polymerases by mainly focusing on their biochemical and structural characteristics, as well as potential protein–protein interactions with other critical factors affecting TLS regulation.     

Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors

Background  

Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved.     

Multifaceted recognition of vertebrate Rev1 by translesion polymerases ζ and κ

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.     

Trading places: how do DNA polymerases switch during translesion DNA synthesis?

The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.     

Regenerating rat liver DNA polymerases: disimilitude or relationship between nuclear and cytoplasmic enzymes?

The possible relationship between the nuclear and cytoplasmic DNA polymerases of regenerating rat liver was studied by sucrose gradient analysis, salt dissociation, and with specific inhibitors. After aqueous subcellular fractionation and removal of the nuclear membranes, three species of DNA-dependent DNA polymerases were characterized: 1) a DNA polymerase-beta in the nuclei. 2) a DNA polymerase-alpha in the cytosol which was not dissociated at high salt concentrations; and 3) an intermediate form in the cytosol and in the Triton wash containing the nuclear membranes. The latter form behaved like DNA polymerase-alpha et low salt concentration but was dissociated at high salt concentrations to a low molecular weight species with properties like DNA polymerase-beta (resistance to inhibition by N-ethylmaleimide, heparin and KCL). In vitro reassociation experiments suggest that this intermediate form corresponds to the association of DNA polymerase-beta with a membrane component or cytoplasmic protein(s) which appear(s) in regenerating rat liver.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.     

Crosstalk between replicative and translesional DNA polymerases: PDIP38 interacts directly with Polη

Replicative DNA polymerases duplicate genomes in a very efficient and accurate mode. However their progression can be blocked by DNA lesions since they are unable to accommodate bulky damaged bases in their active site. In response to replication blockage, monoubiquitination of PCNA promotes the switch between replicative and specialized polymerases proficient to overcome the obstacle. In this study, we characterize novel connections between proteins involved in replication and TransLesion Synthesis (TLS). We demonstrate that PDIP38 (Polδ interacting protein of 38 kDa) directly interacts with the TLS polymerase Polη. Interestingly, the region of Polη interacting with PDIP38 is found to be located within the ubiquitin-binding zinc finger domain (UBZ) of Polη. We show that the depletion of PDIP38 increases the number of cells with Polη foci in the absence of DNA damage and diminishes cell survival after UV irradiation. In addition, PDIP38 is able to interact directly not only with Polη but also with the specialized polymerases Rev1 and Polζ (via Rev7). We thus suggest that PDIP38 serves as a mediator protein helping TLS Pols to transiently replace replicative polymerases at damaged sites.     

DNA polymerases and SOS mutagenesis: can one reconcile the biochemical and genetic data?

Until recently, it had been concluded from genetic evidence that DNA polymerase III (Pol III, the main replicative polymerase in E. coli) was also responsible for mutagenic translesion synthesis on damaged templates, albeit under the influence of inducible proteins UmuD' and UmuC. Now it appears that these proteins themselves have polymerase activity (and are now known as Pol V) and can carry out translesion synthesis in vitro in the absence of Pol III. Here I discuss the apparent contradictions between genetics and biochemistry with regard to the role of Pol III in translesion synthesis. Does Pol V interact with Pol III and constitute an alternative component of the replication factory (replisome)? Where do the other three known polymerases fit in? What devices does the cell have to ensure that the "right" polymerase is used in a given situation? The debate about the role of Pol III in translesion synthesis reveals a deeper divide between models that interpret everything in terms of mass action effects and those that embrace a replisome held together by protein-protein interactions and located as a structural entity within the cell.     

Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.     

A one and a two … expanding roles for poly(ADP-ribose) polymerases in metabolism

In two related papers in this issue, Bai et al. (2011a, 2011b) observe that PARP-1- or PARP-2-deficient mice show increased energy expenditure and protection against diet-induced obesity. This metabolic phenotype is achieved, in part, through SIRT1 activation, either by increasing NAD(+) levels or by promoting SIRT1 expression.     

DNA polymerases ν and θ are required for efficient immunoglobulin V gene diversification in chicken

The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis–dependent point mutations (Ig hypermutation) and homologous recombination (HR)–dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polν and Polθ led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN^−/−/POLQ^−/− cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Polη has also been previously implicated in Ig gene conversion. We show that a POLH^−/−/POLN^−/−/POLQ^−/− triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polν and Polθ in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.     

Critical amino acids in human DNA polymerases η and κ involved in erroneous incorporation of oxidized nucleotides

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Polη and Polκ proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Polη was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Polη) to 7 : 1 (R61K). Similarly, Tyr112 in Polκ was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polκ) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Polη, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Polη and Polκ might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.