Letter to the Editor Letter to the Editor amino acid sequence identity and similarity amongst them-The Y-Family of DNA Polymerases UmuC/DinB/Rev1/Rad30 DNA polymerases be referred ESBS Boulevard S. Brant to as the "Y-family" of DNA polymerases.Phylogenetic analysis of the Y-family of DNA poly-67400 Strasbourg France merases reveals several branches to an unrooted tree (Figure 1). Interestingly, not all branches are evenly dis-
Devices that convert information from one form into another according to a definite procedure are known as automata. One such hypothetical device is the universal Turing machine, which stimulated work leading to the development of modern computers. The Turing machine and its special cases, including finite automata, operate by scanning a data tape, whose striking analogy to information-encoding biopolymers inspired several designs for molecular DNA computers. Laboratory-scale computing using DNA and human-assisted protocols has been demonstrated, but the realization of computing devices operating autonomously on the molecular scale remains rare. Here we describe a programmable finite automaton comprising DNA and DNA-manipulating enzymes that solves computational problems autonomously. The automaton's hardware consists of a restriction nuclease and ligase, the software and input are encoded by double-stranded DNA, and programming amounts to choosing appropriate software molecules. Upon mixing solutions containing these components, the automaton processes the input molecule via a cascade of restriction, hybridization and ligation cycles, producing a detectable output molecule that encodes the automaton's final state, and thus the computational result. In our implementation 1012 automata sharing the same software run independently and in parallel on inputs (which could, in principle, be distinct) in 120 microl solution at room temperature at a combined rate of 109 transitions per second with a transition fidelity greater than 99.8%, consuming less than 10-10 W.
Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD, RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD, RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10 -100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase .Mutagenesis caused by UV light and by many other DNA damaging agents in Escherichia coli is under control of the SOS response, a highly regulated stress response, which functions to increase cell survival under adverse environmental conditions that cause DNA damage (1). Genetic analysis has uncovered four genes, whose products are required for SOS mutagenesis. Two of these, DNA polymerase III (pol-III) 1 and RecA, participate also in replication and recombination, respectively.The other two, UmuD and UmuC, are specifically required for the mutagenic reaction. It was found that UmuD is processed into a shorter form, UmuDЈ, which is the form active in SOS mutagenesis (reviewed in Ref.2).Based on in vivo and in vitro data, UmuDЈ and UmuC were thought to be accessory proteins, which assist DNA polymerase III in replicating DNA lesions which usually block replication (2-5). According to this mechanism, the mutations occur by misinsertion opposite the DNA lesion by the DNA polymerase, a result of the miscoding nature of most DNA lesions. Recently SOS mutagenesis was reconstituted with purified components in two laboratories (6, 7). The results, which confirmed an earlier study (4), provided strong biochemical evidence that SOS mutagenesis occurs by replication through DNA lesions, in a reaction which depends on UmuC, UmuDЈ, RecA and SSB. Moreover, it was shown that there is a qualitative difference in the specificity of bypass when translesion replication was compared in the absence or presence of SOS proteins. DNA polymerase III holoenzyme bypassed an abasic site via a misalignment mechanism, resulting in skipping over the lesion, and the formation of Ϫ1 frameshifts (7,8). In contrast, in the p...
DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error-free, and the third slow and error-prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase f (polf), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two-polymerase combinations with polf dictate error-prone or error-free TLS across the same lesion. These results highlight the central role of polf in both error-prone and error-free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two-polymerase combinations.
Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK ؉/؉ mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 ؎ 4%, which is an order of magnitude higher than in Escherichia coli. In PolK ؊/؊ MEFs, bypass was 16 ؎ 1%, suggesting that at least twothirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase (pol ). In contrast, pol was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking pol . Expression of pol from a plasmid introduced into PolK ؊/؊ MEFs restored both the extent and fidelity of bypass across BP-G. Pol was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol and pol , suggesting that the biological role of pol in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that pol bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.Genomic DNA is constantly subject to damage caused by both external agents, such as sunlight, and endogenous chemicals, such as reactive oxygen species. Most of this damage is eliminated by error-free DNA repair mechanisms, thereby restoring the DNA to its native sequence (1). However, a significant number of lesions escape repair and might therefore interfere with DNA replication and gene expression. Such interference can be mitigated by DNA damage tolerance mechanisms, primarily translesion DNA synthesis (TLS 1 ; also termed translesion replication) (2-5) and postreplicative recombinational repair (1, 6 -8). The key components in TLS are low fidelity DNA polymerases that specialize in lesion bypass (9 -12). These proteins were conserved in evolution and are present in organisms ranging from Escherichia coli to humans (13). Humans contain at least four specialized DNA polymerases belonging to the Y superfamily (pol , pol , pol , and REV1) as well as several from other polymerase families (e.g. pol (14), pol (15, 16), and pol (16, 17)). Many of these polymerases have been implicated in TLS in vitro (18 -24). However, there is a paucity of information about the efficiency and fidelity with which they support lesion bypass in living cells.Pol has a well established biological role in TLS, since it is mutated in all patients examined with the variant form of the hereditary disease xeroderma pigmentosum (10,18). This disease is characterized by sensitivi...
Background: An Israeli national taskforce performed a multi-center clinical and analytical validation of seven serology assays to determine their utility and limitations for SARS-CoV-2 diagnosis. Methods: Serology assays from Roche, Abbott, Diasorin, BioMerieux, Beckman-Coulter, Siemens, and Mt.-Sinai ELISA were included. Negative samples from 2391 individuals representative of the Israeli population, and 698 SARS-CoV-2 PCR positive patients, collected between March and May 2020, were analyzed Findings: Immunoassays sensitivities between 81.5%-89.4% and specificities between 97.7%-100% resulted in a profound impact on the expected Positive Predictive Value (PPV) in low (<15%) prevalence scenarios. No meaningful increase was detected in the false positive rate in children compared to adults. A positive correlation between disease severity and antibody titers, and no decrease in antibody titers in the first 8 weeks after PCR positivity was observed. We identified a subgroup of symptomatic SARS-CoV-2 positive patients (~5% of patients), who remained seronegative across a wide range of antigens, isotypes, and technologies. Interpretation: wThe commercially available automated immunoassays exhibit significant differences in performance and expected PPV in low prevalence scenarios. The low false-positivity rate in under 20 0 s suggests that cross-reactive immunity from previous CoV strains is unlikely to explain the milder disease course in children. Finding no decrease in antibody titers in the first 8 weeks is in contrast to some reports of short half-life for SARS-CoV-2 antibodies. The~5% who were seronegative non-responders, using multiple
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