The length of telomeric G-rich strand 3′-overhang measured by oligonucleotide ligation assay

A typical G-rich telomeric DNA strand, which runs 5′→3′ toward the chromosome ends, protrudes by several nucleotides in lower eukaryotes. In human chromosomes long G-rich 3′-overhangs have been found. Apart from the standard G-rich tail, several non-canonical terminal structures have been proposed. However, the mechanism of long-tail formation, the presence and the role of these structures in telomere maintenance or shortening are not completely understood. In a search for a simple method to accurately measure the 3′-overhang we have established a protocol based on the ligation of telomeric oligonucleotide hybridized to non-denatured DNA under stringent conditions (oligonucleotide ligation assay with telomeric repeat oligonucleotide). This method enabled us to detect a large proportion of G-rich single-stranded telomeric DNA that was as short as 24 nt. Nevertheless, we showed G-tails longer than 400 nt. In all tested cells the lengths ranging from 108 to 270 nt represented only 37% of the whole molecule population, while 56–62% were <90 nt. Our protocol provides a simple and sensitive method for measuring the length of naturally occurring unpaired repeated DNA.

C/EBPɛ mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut)

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBPɛ) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBPɛ activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBPɛ−/− mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBPɛ and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBPɛ in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBPɛ mRNA. We therefore hypothesize that C/EBPɛ positively regulates SGP gene expression, and that C/EBPɛ is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBPɛ promoter is regulated by CDP/cut during myeloid differentiation.

Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G0 fibroblasts were either preheated (45°C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 × 10–3 dsb/Gy/Mb. Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12–16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular.

Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation

Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability. The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction. Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro. Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork. Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm. Thus, RecG translocates simultaneously along two DNA strands, one with 5′-3′ and the other with 3′-5′ polarity. The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart. Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.

Single-strand interruptions in replicating chromosomes cause double-strand breaks

Replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template DNA and collapse, generating double-strand ends. To model replication fork collapse in vivo, I constructed phage λ chromosomes carrying the nicking site of M13 bacteriophage and infected with these substrates Escherichia coli cells, producing M13 nicking enzyme. I detected double-strand breaks at the nicking sites in λ DNA purified from these cells. The double-strand breakage depends on (i) the presence of the nicking site; (ii) the production of the nicking enzyme; and (iii) replication of the nick-containing chromosome. Replication fork collapse at nicks in template DNA explains diverse phenomena, including eukaryotic cell killing by DNA topoisomerase inhibitors and inviability of recombination-deficient vertebrate cell lines.

The tight linkage between DNA replication and double-strand break repair in bacteriophage T4

Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.

Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange

Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins.

Inter-strand C-H...O hydrogen bonds stabilizing four-stranded intercalated molecules: stereoelectronic effects of O4' in cytosine-rich DNA.

DNA fragments with stretches of cytosine residues can fold into four-stranded structures in which two parallel duplexes, held together by hemiprotonated cytosine.cytosine+ (C.C+) base pairs, intercalate into each other with opposite polarity. The structural details of this intercalated DNA quadruplex have been assessed by solution NMR and single crystal x-ray diffraction studies of cytosine-rich sequences, including those present in metazoan telomeres. A conserved feature of these structures is the absence of stabilizing stacking interactions between the aromatic ring systems of adjacent C.C+ base pairs from intercalated duplexes. Effective stacking involves only the exocyclic keto groups and amino groups of the cytidine bases. The apparent absence of stability provided by stacking interactions between the bases in this intercalated DNA has prompted us to examine the available structures in detail, in particular with regard to unusual features that could compensate for the lack of base stacking. In addition to base-on-deoxyribose stacking and intra-cytidine C-H...O hydrogen bonds, this analysis reveals the presence of a hitherto unobserved, systematic intermolecular C-H...O hydrogen bonding network between the deoxyribose sugar moieties of antiparallel backbones in the four-stranded molecule.

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.

Negative-strand RNA viruses: genetic engineering and applications.

The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.

The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound to the RecA protein primary site. We conclude that the specificity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNa molecules within nucleoprotein filament--i.e., self-recognition. The secondary DNA binding site displays a higher affinity for ssDNA than for double-stranded DNA, and the binding of ssDNA to the secondary site strongly inhibits DNA strand exchange. We suggest that the secondary binding site has a dual role in DNA strand exchange. During the homology search, it binds double-stranded DNA weakly; upon finding local homology, this site binds, with higher affinity, the ssDNA strand that is displaced during DNA strand exchange. These characteristics facilitate homologous pairing, promote stabilization of the newly formed heteroduplex DNA, and contribute to the directionality of DNA strand exchange.