Intrinsic coupling of lagging-strand synthesis to chromatin assembly

Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis--to our knowledge--of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.     

DNA primase acts as a molecular brake in DNA replication

A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.     

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.     

Human DNA ligase I completely encircles and partially unwinds nicked DNA

The end-joining reaction catalysed by DNA ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized mammalian DNA ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5' adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein-protein interface that may coordinate the joining of Okazaki fragments.     

利用pGenesil-1.0构建人Dna2真核表达干扰载体

目的:构建人基因Dna2干扰(RNAi)的真核表达载体.方法:根据GenBank上人Dna2的cDNA序列设计并合成两条单链寡核苷酸,退火以后,插入质粒pGenesil-1.0多克隆位点的BamH I和Hind III之间进行重组,构建重组干扰载体,转化DH5α后测序鉴定出阳性菌株.重组质粒以脂质体法转染HeLa细胞,提取Dna2蛋白以免疫印迹法检验干扰效果.结果:设计的目的干扰序列5’-catagccagtagtattcgatg-3’可明显抑制Dna2在HeLa细胞的表达.结论:利用pGenesil-1.0构建的人Dna2干扰载体适用于该基因功能研究.     

Crystal structure of the specificity domain of ribonuclease P

RNase P is the only endonuclease responsible for processing the 5' end of transfer RNA by cleaving a precursor and leading to tRNA maturation. It contains an RNA component and a protein component and has been identified in all organisms. It was one of the first catalytic RNAs identified and the first that acts as a multiple-turnover enzyme in vivo. RNase P and the ribosome are so far the only two ribozymes known to be conserved in all kingdoms of life. The RNA component of bacterial RNase P can catalyse pre-tRNA cleavage in the absence of the RNase P protein in vitro and consists of two domains: a specificity domain and a catalytic domain. Here we report a 3.15-A resolution crystal structure of the 154-nucleotide specificity domain of Bacillus subtilis RNase P. The structure reveals the architecture of this domain, the interactions that maintain the overall fold of the molecule, a large non-helical but well-structured module that is conserved in all RNase P RNA, and the regions that are involved in interactions with the substrate.     

Repetitive shuttling of a motor protein on DNA

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.     

Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase

Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function. Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication. DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication. However, the template-directed copying of DNA is identical in all cases. The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA. The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E. coli. We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor. The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases.     

An interferon-induced cellular enzyme is incorporated into virions

The mechanisms by which interferon inhibits viral growth are only partially understood. Several enzymatic activities increase in cells shortly after treatment with interferon. One of these enzymes, oligo-isoadenylate synthetase, synthesizes (2'-5') isoadenylate oligomers which strongly stimulate the activity of a cellular ribonuclease, RNase F (ref. 7). Interferon also significantly increases the activity of a protein kinase which phosphorylates the initiation factor eIF-2 and can inhibit in vitro protein synthesis. Such interferon-induced enzymes, which affect RNA and protein metabolism, might be responsible for many of its effects on viruses. Indeed, inhibition of viral protein and RNA synthesis appears to have a major role in the antiviral state. We have now investigated possible interactions of the two enzymes with viral constituents during the course of infection and found that in two different membrane-coated RNA viruses, vesicular stomatitis virus (VSV) and Moloney murine leukaemia virus (M-MuLV), there is an accumulation of the 2'-5') oligo-isoadenylate synthetase (E) in the virions. Most of the enzyme is bound to the virion ribonucleoprotein core. The incorporation of E into the virions suggests a direct involvement of the enzyme in regulation of virus functions.     

Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA

Heat-shock proteins are normal constituents of cells whose synthesis is increased on exposure to various forms of stress. They are interesting because of their ubiquity and high conservation during evolution. Two families of heat-shock proteins, hsp60s and hsp70s, have been implicated in accelerating protein folding and oligomerization and also in maintaining proteins in an unfolded state, thus facilitating membrane transport. The Escherichia coli hsp70 analogue, DnaK, and two other heat-shock proteins, DnaJ and GrpE, are required for cell viability at high temperatures and are involved in DNA replication of phage lambda and plasmids P1 and F. These three proteins are involved in replication in vitro of P1 DNA along with many host replication proteins and the P1 RepA initiator protein. RepA exists in a stable protein complex with DnaJ containing a dimer each of RepA and DnaJ. We report here that DnaK and DnaJ mediate an alteration in the P1 initiator protein, rendering it much more active for oriP1 DNA binding.     

RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 +/- 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 +/- 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.     

Infective viruses produced from full-length complementary DNA of swine vesicular disease viruses HK/70 strain

Swine vesicular disease (SVD) is a highly conta- gious viral disease of pigs. Symptoms are clinically indistinguishable from those caused by other vesiculardisease viruses, such as foot and mouth disease (FMD virus, vesicular stomatitis (VS) virus and ves…     

马铃薯卷叶病毒基因组保守序列片段的RT—PCR扩增

从感染马铃薯卷叶病毒的植株中提取马铃薯卷叶病毒总RNA,针对马铃薯卷叶病毒基因组,其中的2个保守序列分别设计并合成了1对寡聚核苷酸引物,用反转录-聚合酶链式反应方法从提取的病毒RNA材料中扩增出符合设计大小的240bp、400bp的特异性产物,对照的健康植株中未扩增出相应产物。