抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Cells utilize diverse ATP-dependent nucleosome-remodelling complexes to carry out histone sliding, ejection or the incorporation of histone variants, suggesting that different mechanisms of action are used by the various chromatin-remodelling complex subfamilies. However, all chromatin-remodelling complex subfamilies contain an ATPase-translocase 'motor' that translocates DNA from a common location within the nucleosome. In this Review, we discuss (and illustrate with animations) an alternative, unifying mechanism of chromatin remodelling, which is based on the regulation of DNA translocation. We propose the 'hourglass' model of remodeller function, in which each remodeller subfamily utilizes diverse specialized proteins and protein domains to assist in nucleosome targeting or to differentially detect nucleosome epitopes. These modules converge to regulate a common DNA translocation mechanism, to inform the conserved ATPase 'motor' on whether and how to apply DNA translocation, which together achieve the various outcomes of chromatin remodelling: nucleosome assembly, chromatin access and nucleosome editing.

Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes

Helicases and remodeling enzymes are ATP-dependent motor proteins that play a critical role in every aspect of RNA and DNA metabolism. Most RNA-remodeling enzymes are members of helicase superfamily 2 (SF2), which includes many DNA helicase enzymes that display similar structural and mechanistic features. Although SF2 enzymes are typically called helicases, many of them display other types of functions, including single-strand translocation, strand annealing, and protein displacement. There are two mechanisms by which RNA helicase enzymes unwind RNA: The nonprocessive DEAD group catalyzes local unwinding of short duplexes adjacent to their binding sites. Members of the processive DExH group often translocate along single-stranded RNA and displace paired strands (or proteins) in their path. In the latter case, unwinding is likely to occur by an active mechanism that involves Brownian motor function and stepwise translocation along RNA. Through structural and single-molecule investigations, researchers are developing coherent models to explain the functions and dynamic motions of helicase enzymes.

Translocation and Unwinding Mechanisms of RNA and DNA Helicases

Advances in genomics technology have provided the means to probe myriad chromatin interactions at unprecedented spatial and temporal resolution. This has led to a profound understanding of nucleosome organization within the genome, revealing that nucleosomes are highly dynamic. Nucleosome dynamics are governed by a complex interplay of histone composition, histone post-translational modifications, nucleosome occupancy and positioning within chromatin, which are influenced by numerous regulatory factors, including general regulatory factors, chromatin remodellers, chaperones and polymerases. It is now known that these dynamics regulate diverse cellular processes ranging from gene transcription to DNA replication and repair.

Understanding nucleosome dynamics and their links to gene expression and DNA replication.

The nature of the nucleosomal barrier that regulates access to the underlying DNA during many cellular processes is not fully understood. Here we present a detailed map of histone-DNA interactions along the DNA sequence to near base pair accuracy by mechanically unzipping single molecules of DNA, each containing a single nucleosome. This interaction map revealed a distinct approximately 5-bp periodicity that was enveloped by three broad regions of strong interactions, with the strongest occurring at the dyad and the other two about +/-40-bp from the dyad. Unzipping up to the dyad allowed recovery of a canonical nucleosome upon relaxation of the DNA, but unzipping beyond the dyad resulted in removal of the histone octamer from its initial DNA sequence. These findings have important implications for how RNA polymerase and other DNA-based enzymes may gain access to DNA associated with a nucleosome.

/pdf/high-resolution-dynamic-mapping-of-histone-dna-interactions-4zv0jd9qoz.pdf

High-resolution dynamic mapping of histone-DNA interactions in a nucleosome

Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.

/pdf/dynamic-regulation-of-transcription-factors-by-nucleosome-3wipl672lz.pdf

Dynamic regulation of transcription factors by nucleosome remodeling

Most helicases — ubiquitous motor proteins that catalyse strand separation of base-paired nucleic acids — use ATP as an energy source. The hexameric helicase of T7 bacteriophage, the gene 4 protein, does not unwind DNA efficiently in the presence of ATP but instead uses deoxythymine triphosphate (dTTP). Using a single-molecule approach, Michelle Wang and colleagues show that with this helicase, ATP allows repeated slips during unwinding that prevent unwinding over any significant distance. This behaviour is not observed with dTTP. Using these two nucleotides, they show that the six subunits act together to coordinate nucleotide binding and hydrolysis in a way that promotes processive unwinding of the DNA. Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination1,2. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5′ to 3′ along single-stranded (ss)DNA2,3,4,5,6. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate1,7. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates Vmax, Michaelis constants KM and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

/pdf/atp-induced-helicase-slippage-reveals-highly-coordinated-3pknhz1ejt.pdf

ATP-induced helicase slippage reveals highly coordinated subunits

Optical trapping is a powerful single molecule technique used to study dynamic biomolecular events, especially those involving DNA and DNA-binding proteins. Current implementations usually involve only one of stretching, unzipping, or twisting DNA along one dimension. To expand the capabilities of optical trapping for more complex measurements would require a multidimensional technique that combines all of these manipulations in a single experiment. Here, we report the development and utilization of such a novel optical trapping assay based on a three-branch DNA construct, termed a "Y structure". This multidimensional assay allows precise, real-time tracking of multiple configura- tional changes. When the Y structure template is unzipped under both force and torque, the force and extension of all three branches can be determined simultaneously. Moreover, the assay is readily compatible with fluorescence, as demonstrated by unzipping through a fluorescently labeled, paused transcription complex. This novel assay thus allows for the visualization and precision mapping of complex interactions of biomechanical events.

/pdf/dna-y-structure-a-versatile-multidimensional-single-molecule-1eyptyaxy1.pdf

Benjamin Y. Smith

Papers

Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase

Dynamic regulation of transcription factors by nucleosome remodeling

ATP-induced helicase slippage reveals highly coordinated subunits

DNA Y structure: a versatile, multidimensional single molecule assay.