Compartmentalization of prokaryotic DNA replication

It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented.     

DNA replication and nuclear architecture

The model of in situ DNA replication provided by immunofluorescence and confocal imaging is compared with observations obtained by electron microscopic studies. Discrepancies between both types of observations call into question the replication focus as a persistent nuclear structure and as a replication entity where DNA replication takes place. Most electron microscopic analyses reveal that replication sites are confined to dispersed chromatin areas at the periphery of condensed chromatin, and the distribution of replication factors exhibits the same localization pattern. Moreover, rapid migration of newly synthesized DNA from the replication sites towards the interior of condensed chromatin regions obviously takes place during S-phase. It implies modifications of replication domains, hardly detectable by fluorescence microscopy. The confrontation of different observations carried out at light microscopic or electron microscopic levels of resolution lead to a conclusion that a combination of in vivo fluorescence analysis with a subsequent ultrastructural investigation performed on the same cells will represent an optimal approach in future studies of nuclear functions in situ.     

In vivo plasma membrane organization: results of biophysical approaches

In the last two decades, various biophysical techniques have been used to investigate the organization of the plasma membrane in live cells. This review describes some of the most important experimental findings and summarizes the characteristics and limitations of a few frequently used biophysical techniques. In addition, the current knowledge about three membrane organizational elements: the membrane-associated cytoskeleton, caveolae and lipid microdomains, is described in detail. Unresolved issues, experimental contradictions and future directions to integrate the variety of experimental data into a revised model of the plasma membrane of eukaryotic cells are discussed in the last section.     

Single molecule studies of DNA mismatch repair

DNA mismatch repair, which involves is a widely conserved set of proteins, is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair.     

Dynamic elements of replication protein A at the crossroads of DNA replication,recombination, and repair

Abstract

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.     

Essential functions of iron-requiring proteins in DNA replication,repair and cell cycle control

Eukaryotic cells contain numerous iron-requiring proteins such as iron-sulfur (Fe-S) cluster proteins, hemoproteins and ribonucleotide reductases (RNRs). These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these ironrequiring proteins and is genetically associated with diseases characterized by DNA repair defects in mammals. Organisms have evolved multi-layered mechanisms to regulate iron balance to ensure genome stability and cell development. This review briefly provides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.     

Eukaryotic error-prone DNA polymerases: The presumed roles in replication, repair, and mutagenesis

A number of error-prone DNA polymerases have been found in various eukaryotes, ranging from yeasts to mammals, including humans. According to partial homology of the primary structure, they are grouped into families B, X, and Y. These enzymes display a high infidelity on an intact DNA template, but they are accurate on a damaged template. Error-prone DNA polymerases are characterized by probabilities of base substitution or frameshift mutations ranging from 10^?3 to 7.5 · 10^?1 in an intact DNA, whereas the spontaneous mutagenesis rate per replicated nucleotide varies between 10^?10 and 10^?12. Low-fidelity polymerases are terminal deoxynucleotidyl transferase (TdT) and DNA polymerases β, ζ, κ, η, ι, λ, μ, and Rev1. The main characteristics of these enzymes are reviewed. None of them exhibits proofreading 3′ → 5′ exonuclease (PE) activity. The specialization of these polymerases consists in their capacity for synthesizing opposite DNA lesions (not eliminated by the numerous repair systems), which is explained by the flexibility of their active centers or a limited ability to express TdT activity. Classic DNA polymerases α, δ, ε, and γ cannot elongate primers with mismatched nucleotides at the 3′-end (which leads to replication block), whereas some specialized polymerases can catalyze this elongation. This is accompanied by overcoming the replication block, often at the expense of an increased mutagenesis rate. How can a cell exist under the conditions of this high infidelity of many DNA polymerase activities? Not all tissues of the body contain a complete set of low-fidelity DNA polymerases, although some of these enzymes are vitally important. In addition, cells “should not allow” error-prone DNA polymerases to work on undamaged DNA. After a lesion on the DNA template is bypassed, the cell should switch over from DNA synthesis catalyzed by specialized polymerases to the synthesis catalyzed by relatively high-fidelity DNA polymerases δ and ? (with an error frequency of 10^?5 to 10^?6) as soon as possible. This is done by forming complexes of polymerase δ or ? with proliferating cell nuclear antigen (PCNA) and replication factors RP-A and RF-C. These highly processive complexes show a greater affinity to correct primers than specialized DNA polymerases do. The fact that specialized DNA polymerases are distributive or weakly processive favors the switching. The fidelity of these polymerases is increased by the PE function of DNA polymerases δ and ε, as well as autonomous 3′ → 5′ exonucleases, which are widespread over the entire phylogenetic tree of eukaryotes. The exonuclease correction decelerates replication in the presence of lesions in the DNA template but increases its fidelity, which decreases the probability of mutagenesis and carcinogenesis.     

A heterochromatin protein 1 (HP1) dimer and a proliferating cell nuclear antigen (PCNA) protein interact in vivo and are parts of a multiprotein complex involved in DNA replication and DNA repair

Heterochromatin protein 1 (HP1) is a small non-histone chromosomal protein known as a dominant suppressor of position-effect variegation and a major component of heterochromatin. Posttranslationally modified HP1, through interaction with protein partners from different groups, can be involved in a number of nuclear processes, including gene activation, chromatin remodeling, replication and DNA repair. Using bimolecular fluorescence complementation assay and live cell imaging, we demonstrate that HP1β and PCNA, a key player in DNA replication, are closely spaced components of a multiprotein complex involved in replication, both in S phase and during DNA repair, and that the functional complex requires formation of an HP1 dimer.     

Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100–200 nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

Two steps forward,one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).     

Ribonucleotides in DNA: Origins,repair and consequences

While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of “DNA” synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans.     

Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair

The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4 h. However, RPA did not reach its maximum accumulation level until 3 h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8 h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.     

细菌RNA结合蛋白Hfq的DNA压缩和复制作用研究进展

RNA结合蛋白(RNA-Binding Protein)Hfq是一种重要的细菌转录后调节因子,之前对Hfq的研究大多集中在该蛋白对小分子非编码RNA(Small Non-Coding RNA,sRNA)和mRNA的作用上.Hfq最典型的功能是促进sRNA与其靶标mRNA碱基配对,在转录后介导对RNA的稳定性和翻译的调控...     

Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination

Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA. Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair, and recombination in Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica and compared them with their Saccharomyces cerevisiae orthologues. We found that proteins belonging to the replication fork and to the nucleotide excision repair pathway were-on the average-more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination. The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs), are conserved as is Mus81p, involved in resolving meiotic recombination intermediates. Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis are also found in C. glabrata, K. lactis, and D. hansenii but not in Y. lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts. In addition, we found that SGS1 and TOP1, respectively, a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C. glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K. lactis. Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy.