A Brownian ratchet for protein translocation including dissociation of ratcheting sites

We study a model for the translocation of proteins across membranes through a nanopore using a ratcheting mechanism. When the protein enters the nanopore it diffuses in and out of the pore according to a Brownian motion. Moreover, it is bound by ratcheting molecules which hinder the diffusion of the protein out of the nanopore, i.e. the Brownian motion is reflected such that no ratcheting molecule exits the pore. New ratcheting molecules bind at rate γ. Extending our previous approach (Depperschmidt and Pfaffelhuber in Stoch Processes Appl 120:901–925, 2010) we allow the ratcheting molecules to dissociate (at rate δ) from the protein (Model I). We also provide an approximate model (Model II) which assumes a Poisson equilibrium of ratcheting molecules on one side of the current reflection boundary. Using analytical methods and simulations we show that the speeds of both models are approximately the same. Our analytical results on Model II give the speed of translocation by means of a solution of an ordinary differential equation. This speed gives an approximation for the time it takes to translocate a protein of given length.     

Y-family DNA polymerases in Escherichia coli

The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Genetic and biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel DNA polymerases with the ability to catalyze synthesis past DNA lesions that otherwise stall replication--a process termed translesion synthesis (TLS). Similar polymerases have been identified in nearly all organisms, constituting a new enzyme superfamily. Although typically viewed as unfaithful copiers of DNA, recent studies suggest that certain TLS polymerases can perform proficient and moderately accurate bypass of particular types of DNA damage. Moreover, various cellular factors can modulate their activity and mutagenic potential.     

Structural diversity of the Y-family DNA polymerases

The Y-family translesion DNA polymerases enable cells to tolerate many forms of DNA damage, yet these enzymes have the potential to create genetic mutations at high rates. Although this polymerase family was defined less than a decade ago, more than 90 structures have already been determined so far. These structures show that the individual family members bypass damage and replicate DNA with either error-free or mutagenic outcomes, depending on the polymerase, the lesion and the sequence context. Here, these structures are reviewed and implications for polymerase function are discussed.     

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.     

Variations on a theme: Eukaryotic Y-family DNA polymerases

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.     

Y-family DNA polymerases respond to DNA damage-independent inhibition of replication fork progression

In Escherichia coli, the Y-family DNA polymerases Pol IV (DinB) and Pol V (UmuD2'C) enhance cell survival upon DNA damage by bypassing replication-blocking DNA lesions. We report a unique function for these polymerases when DNA replication fork progression is arrested not by exogenous DNA damage, but with hydroxyurea (HU), thereby inhibiting ribonucleotide reductase, and bringing about damage-independent DNA replication stalling. Remarkably, the umuC122::Tn5 allele of umuC, dinB, and certain forms of umuD gene products endow E. coli with the ability to withstand HU treatment (HUR). The catalytic activities of the UmuC122 and DinB proteins are both required for HUR. Moreover, the lethality brought about by such stalled replication forks in the wild-type derivatives appears to proceed through the toxin/antitoxin pairs mazEF and relBE. This novel function reveals a role for Y-family polymerases in enhancing cell survival under conditions of nucleotide starvation, in addition to their established functions in response to DNA damage.     

Y-family DNA polymerases and their role in tolerance of cellular DNA damage

The past 15 years have seen an explosion in our understanding of how cells replicate damaged DNA and how this can lead to mutagenesis. The Y-family DNA polymerases lie at the heart of this process, which is commonly known as translesion synthesis. This family of polymerases has unique features that enable them to synthesize DNA past damaged bases. However, as they exhibit low fidelity when copying undamaged DNA, it is essential that they are only called into play when they are absolutely required. Several layers of regulation ensure that this is achieved.     

Diverse roles of RAD18 and Y-family DNA polymerases in tumorigenesis

Mutagenesis is a hallmark and enabling characteristic of cancer cells. The E3 ubiquitin ligase RAD18 and its downstream effectors, the ‘Y-family’ Trans-Lesion Synthesis (TLS) DNA polymerases, confer DNA damage tolerance at the expense of DNA replication fidelity. Thus, RAD18 and TLS polymerases are attractive candidate mediators of mutagenesis and carcinogenesis. The skin cancer-propensity disorder xeroderma pigmentosum-variant (XPV) is caused by defects in the Y-family DNA polymerase Pol eta (Polη). However it is unknown whether TLS dysfunction contributes more generally to other human cancers. Recent analyses of cancer genomes suggest that TLS polymerases generate many of the mutational signatures present in diverse cancers. Moreover biochemical studies suggest that the TLS pathway is often reprogrammed in cancer cells and that TLS facilitates tolerance of oncogene-induced DNA damage. Here we review recent evidence supporting widespread participation of RAD18 and the Y-family DNA polymerases in the different phases of multi-step carcinogenesis.     

Distinctive genetic features exhibited by the Y-family DNA polymerases in Bacillus subtilis

Translesional DNA polymerases form a large family of structurally related proteins, known as the Y-polymerases. Bacillus subtilis encodes two Y-polymerases, referred herewith as Pol Y1 and Pol Y2. Pol Y1 was expressed constitutively and did not mediate UV mutagenesis. Pol Y1 overexpression increased spontaneous mutagenesis. This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the beta-clamp, and did not require the presence of the RecA protein. In addition, Pol Y1 overexpression delayed cell growth at low temperature. The growth delay was mediated by Pol Y1 interaction with the beta-clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the beta-clamp. In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV-induced mutagenesis was abolished. Upon Pol Y2 overproduction, both UV-induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity. However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the beta-clamp whereas spontaneous mutagenesis did. In addition, Pol Y2-mediated spontaneous mutagenesis required the presence of RecA. Together, these results show that the regulation and the genetic requirements of the two B. subtilis Y-polymerases are different, indicating that they fulfil distinct biological roles. Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E. coli UmuD-related function in growth delay. Pol Y2 exhibits an E. coli Pol V-like mutator activity, but probably acts as a single polypeptide to bypass UV lesions. Thus, B. subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E. coli Y-polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic material.     

Coarse-grained Brownian ratchet model of membrane protrusion on cellular scale

Membrane protrusion is a mechanochemical process of active membrane deformation driven by actin polymerization. Previously, Brownian ratchet (BR) was modeled on the basis of the underlying molecular mechanism. However, because the BR requires a priori load that cannot be determined without information of the cell shape, it cannot be effective in studies in which resultant shapes are to be solved. Other cellular-scale models describing the protrusion have also been suggested for modeling a whole cell; however, these models were not developed on the basis of coarse-grained physics representing the underlying molecular mechanism. Therefore, to express the membrane protrusion on the cellular scale, we propose a novel mathematical model, the coarse-grained BR (CBR), which is derived on the basis of nonequilibrium thermodynamics theory. The CBR can reproduce the BR within the limit of the quasistatic process of membrane protrusion and can estimate the protrusion velocity consistently with an effective elastic constant that represents the state of the energy of the membrane. Finally, to demonstrate the applicability of the CBR, we attempt to perform a cellular-scale simulation of migrating keratocyte in which the proposed CBR is used for the membrane protrusion model on the cellular scale. The results show that the experimentally observed shapes of the leading edge are well reproduced by the simulation. In addition, The trend of dependences of the protrusion velocity on the curvature of the leading edge, the temperature, and the substrate stiffness also agreed with the other experimental results. Thus, the CBR can be considered an appropriate cellular-scale model to express the membrane protrusion on the basis of its underlying molecular mechanism.     

A reexamination of the nucleotide incorporation fidelity of DNA polymerases

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.     

Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases

4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy.     

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5 °C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔH_binding) crosses zero (T_H) and becomes favorable (negative). This T_H/activity upshift temperature is 15 °C for Klenow and 30 °C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG^‡, ΔH^‡, and TΔS^‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp^‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH^‡) switches from positive to negative.     

Role of Y-family translesion DNA polymerases in replication stress: Implications for new cancer therapeutic targets

DNA replication stress, defined as the slowing or stalling of replication forks, is considered an emerging hallmark of cancer and a major contributor to genomic instability associated with tumorigenesis (Macheret and Halazonetis, 2015). Recent advances have been made in attempting to target DNA repair factors involved in alleviating replication stress to potentiate genotoxic treatments. Various inhibitors of ATR and Chk1, the two major kinases involved in the intra-S-phase checkpoint, are currently in Phase I and II clinical trials [2]. In addition, currently approved inhibitors of Poly-ADP Ribose Polymerase (PARP) show synthetic lethality in cells that lack double-strand break repair such as in BRCA1/2 deficient tumors [3]. These drugs have also been shown to exacerbate replication stress by creating a DNA-protein crosslink, termed PARP ‘trapping’, and this is now thought to contribute to the therapeutic efficacy. Translesion synthesis (TLS) is a mechanism whereby special repair DNA polymerases accommodate and tolerate various DNA lesions to allow for damage bypass and continuation of DNA replication (Yang and Gao, 2018). This class of proteins is best characterized by the Y-family, encompassing DNA polymerases (Pols) Kappa, Eta, Iota, and Rev1. While best studied for their ability to bypass physical lesions on the DNA, there is accumulating evidence for these proteins in coping with various natural replication fork barriers and alleviating replication stress. In this mini-review, we will highlight some of these recent advances, and discuss why targeting the TLS pathway may be a mechanism of enhancing cancer-associated replication stress. Exacerbation of replication stress can lead to increased genome instability, which can be toxic to cancer cells and represent a therapeutic vulnerability.     

Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.