Simultaneous Disruption of Two DNA Polymerases,Polη and Polζ, in Avian DT40 Cells Unmasks the Role of Polη in Cellular Response to Various DNA Lesions

Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη^−/−/POLζ^−/− cells from the chicken DT40 cell line. POLζ^−/− cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη^−/−/POLζ^−/− cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ^−/− cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.     

Characteristics and processing of Pol Ⅳ-dependent transcripts in Arabidopsis

<正>In plants,RNA-directed DNA methylation(RdDM)plays an essential role in silencing transposable elements that would otherwise have a deleterious effect on genome integrity.RdDM is an important pathway that establishes and maintains de novo DNA methylation in all three sequence contexts:CG,CHG,and CHH(where H is A,C,or T),and the methylation is targeted by 24-nt     

Translesion DNA polymerases Pol ζ, Pol η, Pol ι, Pol κ and Rev1 are not essential for repeat-induced point mutation inNeurospora crassa

Pol ζ, Pol η, Pol ι, Pol κ and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) inNeurospora crassa. Additionally, we demonstrate that theupr-1 gene, which encodes the catalytic subunit of Pol ζ, is a highly polymorphic locus in Neurospora. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession Nos DQ 231523, DQ 231524, DQ 235021, DQ 235525-DQ 235541, DQ 240287, DQ 240288, DQ 354228, DQ 354235-DQ 354237, DQ 386416-DQ 386422, DQ 387872, DQ494492-DQ494503 and DQ417211-DQ417220.     

A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Polμ and Polλ

Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3′ single-stranded overhangs and trans-polymerization using one 3′ end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence (‘brooch’), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr¹⁴¹, Ala¹⁴², Cys¹⁴³, Gln¹⁴⁴ and Arg¹⁴⁵ in Polµ, and by Trp²³⁹, Val²⁴⁰, Cys²⁴¹, Ala²⁴² and Gln²⁴³ in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.     

Flexible tethering of primase and DNA Pol α in the eukaryotic primosome

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA–DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA–DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.     

The DNA Pol ϵ stimulatory activity of Mrc1 is modulated by phosphorylation

DNA replication checkpoint (Mec1-Mrc1-Rad53 in budding yeast) is an evolutionarily conserved surveillance system to ensure proper DNA replication and genome stability in all eukaryotes. Compared to its well-known function as a mediator of replication checkpoint, the exact role of Mrc1 as a component of normal replication forks remains relatively unclear. In this study, we provide in vitro biochemical evidence to support that yeast Mrc1 is able to enhance the activity of DNA polymerase ? (Pol ?), the major leading strand replicase. Mrc1 can selectively bind avidly to primer/template DNA bearing a single-stranded region, but not to double-stranded DNA (dsDNA). Mutations of the lysine residues within basic patch 1 (BP1) compromise both DNA binding and polymerase stimulatory activities. Interestingly, Mrc1-3D, a mutant mimicking phosphorylation by the Hog1/MAPK kinase during the osmotic stress response, retains DNA binding but not polymerase stimulation. The stimulatory effect is also abrogated in Mrc1 purified from cells treated with hydroxyurea (HU), which elicits replication checkpoint activation. Taken together with previous findings, these results imply that under unperturbed condition, Mrc1 has a DNA synthesis stimulatory activity, which can be eliminated via Mrc1 phosphorylation in response to replication and/or osmotic stresses.     

DNA聚合酶Polι的研究进展

Y家族DNA聚合酶是一种跨损伤复制酶,即能以损伤的DNA为模板进行复制。Y家族DNA聚合酶广泛分布生物界,人类细胞中Y家族DNA聚合酶至少包括Rev1、Polκ、Polι、Polη四种,Polι在以DNA为模板进行复制时错配率很高而不同于其他跨损伤DNA聚合酶,Polι是目前发现的所有DNA聚合酶中保真性最低的DNA聚合酶。很高的错配率导致很高的突变率,最后基因的突变导致癌症的发生,因此Polι在各个国家被广泛的研究,并且对Polι的各个不同的特性进行了研究,取得了一系列成果,现对Polι的研究进展予以综述,并展望了未来的研究趋势。     

DNA聚合酶Polι的研究进展

Y家族DNA聚合酶是一种跨损伤复制酶,即能以损伤的DNA为模板进行复制。Y家族DNA聚合酶广泛分布生物界,人类细胞中Y家族DNA聚合酶至少包括Rev1、Polκ、Polι、Polη四种,Polι在以DNA为模板进行复制时错配率很高而不同于其他跨损伤DNA聚合酶,Polι是目前发现的所有DNA聚合酶中保真性最低的DNA聚合酶。很高的错配率导致很高的突变率,最后基因的突变导致癌症的发生,因此Polι在各个国家被广泛的研究,并且对Polι的各个不同的特性进行了研究,取得了一系列成果,现对Polι的研究进展予以综述,并展望了未来的研究趋势。     

Coupled Van der Pol oscillators — A model of excitatory and inhibitory neural interactions

A system of mutually coupled Van der Pol equations is derived from an extended version of the Wilson and Cowan model for the dynamics of a number of excitatory and inhibitory neural subsets. In the lowest order of approximation, interactions between excitatory and inhibitory subsets appear as linear elastic coupling, while those within and between excitatory and excitatory subsets appear as nonlinear frictional coupling. The case of two coupled oscillators is investigated by the method of averaging and the stability conditions for two mode oscillations are obtained. Internal resonance is also discussed briefly in the case of identical oscillators.     

DNA damage induced Pol η recruitment takes place independently of the cell cycle phase

When DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass. Intriguingly, recent evidence has linked TLS polymerases to processes that can also take place outside S phase such as nucleotide excision repair (NER). Here we show that Pol η is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G1. This observation was confirmed by different strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. The potential connection between Pol η recruitment to DNA lesions outside S phase and NER was further evaluated. Interestingly, the recruitment of Pol η to damage sites outside S phase did not depend on active NER, as UV-induced focus formation occurred normally in XPA, XPG and XPF deficient fibroblasts. Our data reveals that the re-localization of the TLS polymerase Pol η to photo-lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.     

Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts

In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

Human Pol ɛ-dependent replication errors and the influence of mismatch repair on their correction

Mutations in human DNA polymerase (Pol) ?, one of three eukaryotic Pols required for DNA replication, have recently been found associated with an ultramutator phenotype in tumors from somatic colorectal and endometrial cancers and in a familial colorectal cancer. Possibly, Pol ? mutations reduce the accuracy of DNA synthesis, thereby increasing the mutational burden and contributing to tumor development. To test this possibility in vivo, we characterized an active site mutant allele of human Pol ? that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias toward mispairs opposite template pyrimidine bases, particularly T•dTTP mispairs. Expression of mutant Pol ? in human cells lacking functional mismatch repair caused an increase in mutation rate primarily due to T•dTTP mispairs. Functional mismatch repair eliminated the increased mutagenesis. The results indicate that the mutant Pol ? causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol ?. Since tumors from familial and somatic colorectal patients arise with Pol ? mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol ? mutation requiring additional factors to promote tumor development.     

Phosphorylation of the p68 subunit of Pol δ acts as a molecular switch to regulate its interaction with PCNA

DNA polymerase delta (Pol δ) is a central enzyme for eukaryotic DNA replication and repair. Pol δ is a complex of four subunits p125, p68, p50, and p12. The functional properties of Pol δ are largely determined by its interaction with its DNA sliding clamp PCNA (proliferating cellular nuclear antigen). The regulatory mechanisms that govern the association of Pol δ with PCNA are largely unknown. In this study, we identified S458, located in the PCNA-interacting protein (PIP-Box) motif of p68, as a phosphorylation site for PKA. Phosphomimetic mutation of S458 resulted in a decrease in p68 affinity for PCNA as well as the processivity of Pol δ. Our results suggest a role of phosphorylation of the PIP-motif of p68 as a molecular switch that dynamically regulates the functional properties of Pol δ.     

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l^-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l^-/- Polh^-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l^-/- Polh^+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.     

哺乳动物跨损伤DNA聚合酶Polκ研究进展

跨损伤DNA合成(translesion DNA synthesis,TLS)是细胞应答DNA损伤时的一种耐受机制,它利用特异的低保真度的DNA聚合酶直接在损伤的对面合成DNA,使复制得以延续.TLS分为无错旁路和易错旁路两种途径,其中易错旁路途径是DNA损伤诱发基因组突变的主要机制.另外TLS也与肿瘤细胞耐药性相关.在体内执行TLS功能的DNA聚合酶主要是DNA聚合酶Y家族的成员,其中包括聚合酶kappa(Polκ).就TLS的特性,哺乳动物Polκ的结构及催化活性、表达及调控、蛋白质相互作用及其在TLS中可能的调控机制和体内功能等方面做一阐述.     

The BRCT domain and the specific loop 1 of human Polμ are targets of Cdk2/cyclin A phosphorylation

Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polμ participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polμ plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polμ could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polμ. We identified Ser¹², Thr²¹ (located in the BRCT domain) and Ser³⁷² (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser³⁷² is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polμ could be regulated in vivo by phosphorylation of the BRCT domain (Ser¹²/Thr²¹) and of Ser³⁷², affecting the function of loop1. Consequently, Polμ’s most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.     

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.     

Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ

Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. Recessive mutations in Pol γ represent nearly half of those reported to date, and they are nearly uniformly distributed along the length of the POLG1 gene (Human DNA Polymerase gamma Mutation Database); the majority of them are linked to the most severe form of POLG syndrome, Alpers-Huttenlocher syndrome. In this report, we assess the structure-function relationships for recessive disease mutations by reviewing existing biochemical data on site-directed mutagenesis of the human, Drosophila and yeast Pol γs, and their homologs from the family A DNA polymerase group. We do so in the context of a molecular model of Pol γ in complex with primer-template DNA, which we have developed based upon the recently solved crystal structure of the apoenzyme form. We present evidence that recessive mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used as a diagnosis-supporting tool to evaluate the pathogenic role of new Pol γ variants.