Chromatin perturbations during the DNA damage response in higher eukaryotes

The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response.     

Nucleotide excision repair in higher eukaryotes: Mechanism of primary damage recognition

Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation, and bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to severe diseases, including some forms of cancer. In view of the broad substrate specificity of NER, it is of interest to study how a certain set of proteins recognizes DNA lesions in contest of a large excess of intact DNA. The review focuses on DNA damage recognition, the key and, as yet, most questionable step of NER. The main models of primary damage recognition and preincision complex assembly are considered. The model of a sequential loading of repair proteins on damaged DNA seems most reasonable in light of the available data.     

The organization, structure, and inheritance of the ER in higher and lower eukaryotes.

The endoplasmic reticulum (ER) is a fundamental organelle required for protein assembly, lipid biosynthesis, and vesicular traffic, as well as calcium storage and the controlled release of calcium from the ER lumen into the cytosol. Membranes functionally linked to the ER by vesicle-mediated transport, such as the Golgi complex, endosomes, vacuoles-lysosomes, secretory vesicles, and the plasma membrane, originate largely from proteins and lipids synthesized in the ER. In this review we will discuss the structural organization of the ER and its inheritance.     

Co-recessive inheritance: a model for DNA repair and other surveillance genes in higher eukaryotes.

The co-recessive inheritance hypothesis proposes that certain recessively inherited diseases require homozygosity and/or hemizygosity for defective alleles at more than one locus simultaneously for the trait to be expressed. Although this hypothesis was originally proposed in the context of defective alleles for genes coding for DNA-repair functions, it need not be limited to this context, and genetic selection pressure may favor this model for genes involved in surveillance of any type. The co-recessive inheritance hypothesis also predicts extremely high carrier frequencies, likely affecting much of the general population, for defective alleles associated with these rare recessive diseases. The model predicts much lower rates of consanguinity between the parents of affected individuals than autosomal recessive inheritance, allowing it to be tested epidemiologically, and recent data suggest that the hypothesis may be valid for some cases of ataxia telangiectasia and xeroderma pigmentosum. The model provides possible explanations for a number of otherwise puzzling findings in several diseases associated with defective DNA repair.     

An efficient protein complex purification method for functional proteomics in higher eukaryotes

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.     

Triggering cell mitosis in higher eukaryotes

Dramatic changes of cell organisation occur at onset of mitosis. Genetic analysis of fission yeast and physiological studies of vertebrate and invertebrate oocytes showed that activation of cyclin B-cdc2 kinase triggers mitosis. Nevertheless, upstream mechanisms responsible for this activation remain largely unknown in somatic cells of higher eukaryotes. This review discusses possible pathways and mechanisms involved in triggering onset of mitosis in such cells, including inhibitory checkpoint mechanisms that detect defects in structural organisation of the cell.     

Initiation of DNA replication in higher eukaryotes

In this review, of problems concerning initiation of DNA replication in higher eukaryotes is discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Nuclear-pore-complex dynamics and transport in higher eukaryotes

Summary The nuclear-pore complex controls the passage of macromolecules to and from the nucleus. It is a complex structure spanning the double-membrane nuclear envelope, consisting of many proteins and structural components. Structurally it consists of a series of stacked rings and associated filaments and a central cylinder which appears to contain the transport channel. Much of the pore complex appears to be dynamic, altering conformationally during transport. We review what is known about pore complex structure and dynamics and attempt to relate this to recent new information on transport pathways and the interactions of transport factors with each other and with components of the nuclear-pore complex.     

Molecular biology of enzyme adaptations in higher eukaryotes

Multicellular eukaryotes have evolved complex homeostatic mechanisms that buffer the majority of their cells from direct interaction with the external environment. Thus, in these organisms long-term adaptations are generally achieved by modulating the developmental profile and tissue specificity of gene expression. Nevertheless, a subset of eukaryotic genes are still involved in direct responses to environmental fluctuations. It is the adaptative responses in the expression of these genes that buffers many other genes from direct environmental effects. Both microevolutionary and macroevolutionary patterns of change in the structure and regulation of such genes are illustrated by the sequences encoding alpha-amylases. The molecular biology and evolution of alpha-amylases in Drosophila and other higher eukaryotes are presented. The amylase system illustrates the effects of both long-term and short-term natural selection, acting on both the structural and regulatory components of a gene--enzyme system. This system offers an opportunity for linking evolutionary genetics to molecular biology, and it allows us to explore the relationship between short-term microevolutionary changes and long-term adaptations.     

MDC1/NFBD1: a key regulator of the DNA damage response in higher eukaryotes

The protein MDC1/NFBD1 contains a forkhead-associated (FHA) domain and two BRCA1 carboxyl-terminal (BRCT) domains. It interacts with several proteins involved in DNA damage repair and checkpoint signalling, and is phosphorylated in response to DNA damage and during mitosis. Upon treatment of cultured human cells with DNA damaging agents, MDC1/NFBD1 translocates to sites of DNA lesions, where it collaborates with other proteins and with phosphorylated histone H2AX to mediate the accumulation of checkpoint and repair factors into nuclear foci. Down-regulation of MDC1/NFBD1 expression levels by small interfering RNA (siRNA) renders cells hyper-sensitive to DNA damaging agents and leads to defects in cell cycle checkpoint activation and apoptosis. Thus, MDC1/NFBD1 appears to be a key regulator of the DNA damage response in mammalian cells.     

Conserved dual-mode gene regulation programs in higher eukaryotes

Recent genomic data analyses have revealed important underlying logics in eukaryotic gene regulation, such as CpG islands (CGIs)-dependent dual-mode gene regulation. In mammals, genes lacking CGIs at their promoters are generally regulated by interconversion between euchromatin and heterochromatin, while genes associated with CGIs constitutively remain as euchromatin. Whether a similar mode of gene regulation exists in non-mammalian species has been unknown. Here, through comparative epigenomic analyses, we demonstrate that the dual-mode gene regulation program is common in various eukaryotes, even in the species lacking CGIs. In cases of vertebrates or plants, we find that genes associated with high methylation level promoters are inactivated by forming heterochromatin and expressed in a context-dependent manner. In contrast, the genes with low methylation level promoters are broadly expressed and remain as euchromatin even when repressed by Polycomb proteins. Furthermore, we show that invertebrate animals lacking DNA methylation, such as fruit flies and nematodes, also have divergence in gene types: some genes are regulated by Polycomb proteins, while others are regulated by heterochromatin formation. Altogether, our study establishes gene type divergence and the resulting dual-mode gene regulation as fundamental features shared in a broad range of higher eukaryotic species.     

Regulation of myosin II during cytokinesis in higher eukaryotes

Cellular myosin II is the principal motor responsible for cytokinesis. In higher eukaryotes, phosphorylation of the regulatory light chain (MLC) of myosin II is a primary means of activating myosin II and is known to be crucial for the execution of cell division. Because signals transmitted by the mitotic spindle coordinate key spatial and temporal aspects of cytokinesis, such signals should ultimately function to activate myosin II. Thus, it follows that identification of regulatory factors involved in MLC phosphorylation should elucidate the nature of spindle-derived regulatory signals and lead to a model for how they control cytokinesis. However, the identity of these upstream molecules remains elusive. This review (which is part of the Cytokinesis series) summarizes current views of the regulatory pathway controlling MLC phosphorylation and features four candidate molecules that are likely immediate upstream myosin regulators. I discuss proposed functions for MLCK, ROCK, citron kinase and myosin phosphatase during cytokinesis and consider the possibility of a link between these molecules and the signals transmitted by the mitotic spindle.     

Overexpression of membrane proteins from higher eukaryotes in yeasts

Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug–target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.