Enzyme-Free Copying of 12 Bases of RNA with Dinucleotides.

The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used RNA as genetic material. How RNA was copied in the absence of enzymes and what sequences were most likely to have supported replication is not clear. Here we show that mixtures of dinucleotides with C and G as bases copy an RNA sequence of up to 12 nucleotides in dilute aqueous solution. Successful enzyme-free copying occurred with in situ activation at 4 °C and pH 6.0. Dimers were incorporated in favor of monomers when both competed as reactants, and little misincorporation was detectable in mass spectra. Simulations using experimental rate constants confirmed that mixed C/G sequences are good candidates for successful replication with dimers. Because dimers are intermediates in the synthesis of longer strands, our results support evolutionary scenarios encompassing formation and copying of RNA strands in enzyme-free fashion.

Circular single-stranded phage M13 DNA is used as a template for complementary strand synthesis in cytosolic extracts from proliferating HeLa cells. DNA synthesis is initiated by one or maximally two priming events and typically leads to covalently closed double-stranded reaction products. When carried out in the presence of the nuclear chromatin assembly factor CAF-1, complementary strand synthesis is accompanied by nucleosome assembly. This novel system is very useful for the study of basic biochemical aspects concerning the assembly of nucleosomes. The activity of CAF-1 completely depends on complementary strand synthesis and acts stoichiometrically to promote the assembly of nucleosomes in a noncooperative manner. Apparently, CAF-1 activity is coupled to DNA synthesis via a structural feature of replicating DNA, most likely its partial single strandedness.

Bacteriophage f1 DNA replication genes: II. The roles of gene V protein and gene II protein in complementary strand synthesis

Nature of the Complementary Strands Synthesized in Vitro upon the Single-Stranded Circular DNA of Bacteriophage øX174 after Ultraviolet Irradiation

LAST year we celebrated the 100th anniversary of the birth of George Beadle. He, with Edward Tatum, in 1941, proposed one of the major conceptual advances in biology in the 20th century: the existence of a one gene–one enzyme relationship (Beadle and Tatum 1941).2 Unfortunately, like many biologists proposing exciting hypotheses, they could not provide the experimental evidence that would prove they were correct. At that time, the existing knowledge about gene and enzyme structure was inadequate to permit the relationship's verification. George Beadle was aware of this and undoubtedly regretted his inability to provide support for their hypothesis. In a retrospective article written for Phage and the Origins of Molecular Biology, Beadle (1966)(p. 30) states that at the Cold Spring Harbor Symposium of 1951 “the number whose faith in one gene–one enzyme remained steadfast could be counted on the fingers of one hand—with a couple of fingers left over.” Why? Why was their proposed basic relationship not accepted? Why could they not provide proof for their hypothesis? Beadle and Tatum's principal difficulty was that in the 1940s we knew relatively little about the chemical nature of genetic material. The prevailing view then was that genetic material might be protein. Existing information on DNA structure suggested that it consisted of repetitive nucleotide sequences. If correct, it would be unlikely that DNA could specify the many different proteins present in each organism. Also, it was not yet firmly established that polypeptides consist of linear sequences of amino acids. Therefore, during the early years following Beadle and Tatum's proposal of the one gene–one enzyme relationship, our understanding of the structural features of genes and enzymes was insufficient to allow formulation of a finite molecular relationship. Advances in the 1950s dealt with most of these issues and suggested a plausible experimental strategy that could be applied in comparing the structural features of a gene with that of its corresponding protein. One of the earliest relevant findings, by Avery et al. (1944), suggested that the pneumococcal transforming principle—presumably the genetic material of the organism—was very likely DNA (see Lederberg 1994). Unfortunately, the significance of this finding was not appreciated initially; questions were raised about the purity of the transforming principle, our understanding of the transformation process was incomplete, and doubts existed that pneumococcal genetic material would be structurally comparable to the genetic material of higher organisms. Somewhat later, in the early 1950s, Al Hershey and Martha Chase presented their isotopic labeling studies with bacteriophage-infected Escherichia coli. Their results provided what was considered convincing evidence that phage genetic material is in fact DNA (Hershey and Chase 1952; see Stahl 1998). Most importantly, in 1953, Jim Watson and Francis Crick described the double-helical structure of DNA (Watson and Crick 1953). Their contribution changed forever how genetic material would be viewed by everyone. The realization that each gene probably consists of a linear sequence of nucleotides prompted subsequent thought on the existence of a genetic code, an essential consideration with a direct bearing on the gene-enzyme relationship. During this period Matt Meselson and Frank Stahl established that DNA is replicated by synthesis of complementary DNA strands, revealing the elegant features of DNA structure that provide for its exact replication (Meselson and Stahl 1958). Also of great importance, Fred Sanger showed that an insulin polypeptide consists of a unique linear sequence of amino acids (Sanger 1952; Stretton 2002). The significance of the contributions of Sanger, Erwin Chargaff, and others are described in an article by Horace Judson (Judson 1993). The discoveries cited, and observations by others supporting their conclusions, redefined the gene–enzyme relationship of Beadle and Tatum as the gene-protein colinearity hypothesis. Despite these advances in the 1950s, the technology that would have allowed either isolation of a gene or determination of its nucleotide sequence was lacking. Furthermore, since the concept of the genetic code was yet to be developed, knowledge of Watson and Crick's double-helical structure of DNA was initially of little help to those wishing to experimentally address the structural relationship between a gene and the enzyme it was proposed to specify. In the late 1950s Seymour Benzer demonstrated that one could construct a linear fine-structure genetic map by crossing mutants with genetically separable alterations in the same genetic region (Benzer 1957). His findings were consistent with the interpretation that a gene consists of a specific linear sequence of nucleotides. At approximately the same time, Vernon Ingram identified the amino acid change in the hemoglobin of humans with sickle cell anemia, and he developed the protein “fingerprinting” technique, a method that could be applied in detecting the single amino acid change in any mutant protein (Ingram 1958; see Ingram 2004). These two advances provided an excellent pair of experimental approaches that could be applied immediately in testing the gene–protein colinearity concept.

The origin of phage G4 DNA complementary strand synthesis (G4oric) consists of three stem-loop structures (stem loops I, II, and III) that have been proposed as a recognition site for primase during primer RNA (pRNA) synthesis (Godson, G. N., Barrell, B. G., Staden, R., and Fiddes, J. C. (1978) Nat. New Biol. 276, 236-247; Fiddes, J. C., Barrell, B. G., and Godson, G. N. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1081-1085; Sims, J., Capon, D., and Dressler, D. (1979) J. Biol. Chem. 254, 12615-12628). It is generally considered that the double-stranded DNA stem-loop structure is not coated with Escherichia coli single-stranded DNA-binding protein (SSB), but is recognized by primase as naked DNA (Kornberg, A., and Baker, J. (1992) DNA Replication, 2nd Ed., p. 280, W. H. Freeman & Co., New York). Using small G4oric single-stranded DNA fragments of various sizes (302, 278, 149, and 100 nucleotides) consisting of the core 100-nucleotide stem-loop region plus differing lengths of 3'- and 5'-flanking sequence as substrates for gel retardation and DNase I and micrococcal nuclease digestion, we show that under conditions of pRNA synthesis, two SSB tetramers bind to the stem-loop structure. With increasing lengths of 5'- and 3'-flanking sequence, more SSB tetramers are added. Regardless of the number of SSB tetramers bound, however, the region of DNA containing the pRNA initiation site is always left accessible to nuclease digestion. In situ copper-phenanthroline footprinting of individual gel shift assembly intermediates shows that on the 302-nucleotide G4oric, the first two SSB tetramers assemble at random, but the addition of more SSB tetramers results in formation of a unique structure. In this structure, SSB tetramers protect both sides of stem loop III plus the intervening region between stem loops III and I, but leave most of stem loop I and the CTG pRNA initiation site accessible to copper-phenanthroline. Primase can only synthesize pRNA when the stem-loop structure is saturated with SSB and presumably in the unique configuration. The G4oric stem-loop structure therefore appears to dictate the phasing of SSB to leave a primase recognition site as free DNA.

REPLICATION OF PHAGE fd DNA WITH PURIFIED PROTEINS

Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication. The replication properties of CTG.CAG, CGG.CCG, and TTC.GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis. The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin. The deletions may be mediated by hairpins formed by these repeat tracts. The distributions of the deletion products of the CTG.CAG and CGG.CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis. Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes. Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates. Small expansions of the CTG.CAG and CGG.CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand. Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication.

Replication of bacteriophage M13: VIII. Differential effects of rifampicin and nalidixic acid on the synthesis of the two strands of M13 duplex DNA

Synthesis of a complementary strand to match the single-stranded, circular, viral (+) DNA strand of phage phi X174 creates a parental duplex circle (replicative form, RF). This synthesis is initiated by the assembly and action of a priming system, called the primosome [Arai, K. & Kornberg, A (1981) Proc. Natl. Acad. Sci. USA 78, 69-73; Arai, K., Low, R. L. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 707-711]. Of the seven proteins that participate in the assembly and function of the primosome, most all of the components remain even after the DNA duplex is completed and covalently sealed. Remarkably, the primosome in the isolated RF obviates the need for supercoiling of RF by DNA gyrase, an action previously considered essential for the site-specific cleavage by gene A protein that starts viral strand synthesis in the second stage of phi X174 DNA replication. Finally, priming of the synthesis of complementary strands on the nascent viral strands to produce many copies of progeny RF utilizes the same primosome, requiring the addition only of prepriming protein i. thus a single primosome, which becomes associated with the incoming viral DNA in the initial stage of replication, may function repeatedly in the initiation of complementary strands at the subsequent stage of RF multiplication. These patterns of phi X174 DNA replication suggest that a conserved primosome also functions in the progress of the replicating fork of the Escherichia coli chromosome, particularly in initiating the synthesis of nascent (Okazaki) fragments.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Multiplication of the duplex, circular, phage phiX174DNA (replicative form, RF) in stage II of the replicative life cycle has been observed with a crude enzyme preparation [Eisenberg et al. (1976) Proc, Natl. Acad, Sci. USA 73, 1594-1597]. This stage has now been partially reconstituted with purified proteins and subdivided into two stages: II(+) and II(-). In stage II(+), viral (+) strand synthesis is carried out by four proteins: the phage-induced, cistron A-dependent protein, rep-dependent protein, DNA unwinding protein, and DNA polymerase III holenzyme. In stage II(-), complementary (-) strand synthesis utilizes the product of stage II(+) as template and the multiprotein system previously identified in the stage I synthesis of a complementary strand on the viral DNA template to produce RF. The multiprotein system includes DNA unwinding protein, proteins i and n, dnaB protein, dnaC protein, dnaG protein, and DNA polymerase III holoenzyme. A discussion of these two separate mechanism for synthesis of (+) and (-) strands suggests that they may account for essentially all the replicative stages in the life cycle of phiX174.

Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity

Mutagenic carcinogens such as alkylating agents, or physical mutagens such as ultra-violet light, induce lesions in DNA. When DNA containing such lesions replicates during S-phase, exchanges often occur between the two daughter molecules (Wolff et al, 1974). At the cytological level this becomes manifest as exchanges between the two sister chromatids of a metaphase chromosome. To visualize these exchanges, however, the cells must be treated so that the two sister chromatids will be different from one another. This can be accomplished by exploiting the fact that DNA is a double molecule that replicates semiconservatively, i.e., it has two complementary polynucleotide strands that separate from one another, and each acts as a template for the synthesis of new complementary strands. This means that each newly synthesized DNA molecule consists of one old polynucleotide strand that is conserved and a brand new strand.KeywordsChinese Hamster Ovary CellSister ChromatidSister Chromatid ExchangeMouse Liver MicrosomeMutagenic CarcinogenThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The solution behaviors and microstructures of poly(N-isopropylacrylamide)x-poly(ethylene oxide)20-poly(propylene oxide)70-poly(ethylene oxide)20-poly(N-isopropylacrylamide)x (PNIPAmx-PEO20-PPO70-PEO20-PNIPAmx or PNIPAmx-P123-PNIPAmx) pentablock terpolymers with various PNIPAm block lengths in dilute and concentrated aqueous solutions were investigated by micro-differential scanning calorimetry (micro-DSC), static and dynamic light scattering (SLS & DLS), and synchrotron small angle X-ray scattering (SAXS). Two lower critical solution temperatures (LCSTs) were observed for PNIPAmx-P123-PNIPAmx pentablock terpolymers in dilute solutions, which corresponded to LCSTs of PPO and PNIPAm blocks, respectively. The LCST of PPO block shifted from 24.4 °C to 29 °C when the length x of PNIPAm block increased from 10 to 97. The LCST of PNIPAm is around 34.5 °C-35.3 °C and less dependent on the block length x. The PNIPAmx-P123-PNIPAmx pentablock terpolymers formed "associate" structures and micelles with hydrophobic PNIPAm and PPO blocks as cores and soluble PEO blocks as coronas in dilute aqueous solutions at 20 °C and 40 °C, respectively, regardless of the relative lengths of PNIPAm, PPO and PEO blocks. The size of "associate" structures of PNIPAmx-P123-PNIPAmx pentablock terpolymers at 20 °C increased with increasing the length of PNIPAm block. The microstructures of PNIPAmx-P123-PNIPAmx hydrogels formed in concentrated aqueous solutions (40 wt%) were strongly dependent on the environmental temperatures and relative lengths of PNIPAm, PPO and PEO blocks as revealed by SAXS. Increasing the length of PNIPAm block weakened the order structures of PNIPAmx-P123-PNIPAmx hydrogels. The microstructures of PNIPAmx-P123-PNIPAmx hydrogels changed from mixed fcc and hex structures for PNIPAm10-P123-PNIPAm10 to isotropic structure for PNIPAm97-P123-PNIPAm97. Increasing temperature led to the transition from mixed hex and fcc structure to pure hex structure for PNIPAm10-P123-PNIPAm10 hydrogel at temperature above the LCSTs.