Replication and recombination of herpes simplex virus DNA

Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.     

Elemental composition of the sub-aquatic liverwort Pellia endiviifolia (Dicks.) Dum. in relation to heavy metal contamination

The concentrations of fifteen elements (B, Na, Mg, Al, P, K, Ca, Ti, Mn, Fe, Cu, Zn, Sr, Cd, Pb) in thalli of the sub-aquatic liverwort Pellia endiviifolia (Dicks.) Dum. collected from seven sites in Japan, were analyzed by inductively coupled plasma atomic emission spectrometer (ICP). The average concentration of major essential elements in the thalli was: 49 600 µg g^–1 K, 9 140 µg g^–1 Na, 6740 µg g^–1 Ca, 3 840 µg g^–1 P, and 3 060 µg g^–1 Mg. Thalli of P. endiviifolia from sites polluted by heavy metals (Cu, Zn, Pb) contained high concentrations of these elements (maxima 0.88% Cu, 0.55% Zn and 0.36% Pb in the older thallus), thus demonstrating the potentiality of this liverwort as an indicator for heavy metal contamination.     

An Iron–Sulfur Cluster in the Polymerase Domain of Yeast DNA Polymerase ε

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits—Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1–1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2^ins) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.     

Reconstructing larval growth and habitat use in an amphidromous goby using otolith increments and microchemistry

High‐resolution analysis of growth increments, trace element chemistry and oxygen isotope ratios (δ¹⁸O) in otoliths were combined to assess larval and post‐larval habitat use and growth of Awaous stamineus, an amphidromous goby native to Hawai‘i. Otolith increment widths indicate that all individuals experience a brief period of rapid growth during early life as larvae and that the duration of this growth anomaly is negatively correlated with larval duration. A protracted high‐growth period early in larval life is associated with a lower ratio of Sr:Ca, which may reflect low salinity conditions in nearshore habitats. A distinct shift in δ¹⁸O (range: 4–5‰) is closely associated with the metamorphic mark in otoliths, indicating that larval metamorphosis occurs promptly upon return to fresh water. Strontium and other trace elements are not as tightly coupled to the metamorphosis mark, but confirm the marine‐to‐freshwater transition. Integration of microstructural and microchemical approaches reveals that larvae vary substantially in growth rate, possibly in association with habitat differences. Although time and financial costs make it difficult to achieve large sample sizes, present results show that examining even a small number of individuals can lead to novel inferences about early life history in diadromous fishes and illustrates the value of integrating analyses.     

Natural variability and effects of cleaning and storage procedures on vertebral chemistry of the blacktip shark Carcharhinus limbatus

Key methodological assumptions regarding the degree of natural variability and influence of sample handling and storage of elasmobranch vertebral chemistry were assessed using laser‐ablation inductively coupled plasma mass spectrometry. Vertebral chemistry of juvenile blacktip sharks Carcharhinus limbatus was examined to identify whether differences existed among different regions of the vertebral column, between thoracic vertebrae of individual fish or within individual vertebrae. Additionally, the effects of bleach exposure and storage in ethanol on vertebral chemistry were compared. No significant variation in vertebral chemistry was found among different regions of the vertebral column or between thoracic vertebrae, but significant differences among transect locations within individual vertebrae were observed. The variation at all three levels appears comparable with published data on sagittal otoliths of bilaterally symmetrical teleost fishes. The experimental assessment of potential treatment effects indicated vertebral chemistry was not significantly affected by bleach or exposure to ethanol. Taken together, these results support the idea that vertebrae taken from the same region of the vertebral column can be treated as equivalent and at least certain elements remain robust to exposure to bleach and ethanol.     

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer''s and Parkinson''s disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.     

两种常见绿化树种对大气颗粒物的滞留与再悬浮

植物叶片对大气颗粒物的作用机制可分为吸附、再悬浮、雨水淋洗、吸收等过程,但却少有研究探讨大气颗粒物沉降于叶片表面后的再悬浮过程,并对叶片表面吸附的颗粒物含量与叶片内部元素含量之间的相关性进行探讨.本研究选择二球悬铃木和广玉兰两种常见绿化树种,分析其叶表大气颗粒物和叶内元素成分与含量及再悬浮比例.结果表明: 二球悬铃木叶片的单位面积滞尘量(4.98 g·m^-2)明显高于广玉兰(2.65 g·m^-2),这可能与二球悬铃木叶片上下表面均粗糙、有绒毛有关.电感耦合等离子体发射光谱(ICP)分析表明,15种不同的元素均可在二球悬铃木和广玉兰叶表和叶内监测出来,在整体上两树种叶表与叶内元素含量间存在明显的相关性.但仅有Cr、Fe、V等 3种元素在二球悬铃木叶表与叶内间存在显著的相关性,而广玉兰中仅有K、Mn、Si、Ti和Zn 等5种元素没有显著的相关性,说明两树种叶片对不同元素的吸收具有较强的选择性.再悬浮分析表明,无论是随着风速的增加还是随着风力作用时间的延长,大气颗粒物的再悬浮比例均随之显著增加.在研究植物对大气颗粒物滞留效果时,必须考虑到颗粒物的再悬浮,才能准确评估植被与大气颗粒物的沉降关系.     

Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nuclease

The replication of herpes simplex virus type 1 (HSV-1) is associated with a high degree of homologous recombination, which is likely to be mediated, in part, by HSV-1-encoded proteins. We have previously shown that the HSV-1 encoded ICP8 protein and alkaline nuclease UL12 are capable of catalyzing an in vitro strand-exchange reaction. Here, we show, by electron microscopy, that the products of the strand exchange reaction between linear double-stranded DNA and circular single-stranded DNA consist of the expected joint molecule forms: sigma, alpha, and gapped circles. Other exonucleases, such as lambda Red alpha, which, like UL12, digests 5'-3', as well as Escherichia coli exonuclease III (ExoIII), which digests 3'-5', could substitute for UL12 in the strand exchange reaction by providing a resected DNA end. ICP8 generated the same intermediates and strand exchange products when the double-stranded DNA substrate was preresected by any of the nucleases. Using substrates with large regions of non-homology we found that pairing by ICP8 could be initiated from the middle of a DNA molecule and did not require a homologous end. In this reaction, the resection of a DNA end by the nuclease is required to reveal homologous sequences capable of being paired by ICP8. This study further illustrates the complexity of the multi-functional ICP8 protein.