A method to monitor replication fork progression in mammalian cells: nucleotide excision repair enhances and homologous recombination delays elongation along damaged DNA

The capacity to rescue stalled replication forks (RFs) is important for the maintenance of cell viability and genome integrity. Here, we have developed a novel method for monitoring RF progression and the influence of DNA lesions on this process. The method is based on the principle that each RF is expected to be associated with a pair of single-stranded ends, which can be analyzed by employing strand separation in alkali. This method was applied to examine the rate of RF progression in Chinese hamster cell lines deficient in ERCC1, which is involved in nucleotide excision repair (NER), or in XRCC3, which participates in homologous recombination repair, following irradiation with ultraviolet (UV) light or exposure to benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE). The endpoints observed were cell survival, NER activity, formation of double-strand breaks and the rate of RF progression. Subsequently, we attempted to explain our observation that cells deficient in XRCC3 (irs1SF) exhibit enhanced sensitivity to UV radiation and BPDE. irs1SF cells demonstrated a capacity for NER that was comparable with wild-type AA8 cells, but the rate of RF progression was even higher than that for the wild-type AA8 cells. As expected, cells deficient in ERCC1 (UV4) showed no NER activity and were hypersensitive to both UV radiation and BPDE. The observation that cells deficient in NER displayed a pronounced delay in RF progression indicates that NER plays an important role in maintaining fork progression along damaged DNA. The elevated rate of RF progression in XRCC3-deficient cells indicates that this protein is involved in a time-consuming process which resolves stalled RFs.     

Mapping rheumatoid factor binding sites using genetically engineered, chimeric IgG antibodies.

We are using chimeric IgG antibodies consisting of murine variable regions joined to human constant regions as rheumatoid factor (RF) binding substrates to localize and map IgM RF binding sites on IgG. Using chimeric antibodies in a modified RF ELISA, we showed that RFs from rheumatoid arthritis (RA) and Waldenstrom's macroglobulinemia (WMac) patients differ in their binding specificities for IgG3, although some of these RFs share common specificity for IgG1, IgG2, and IgG4. By shuffling constant region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for WMac-derived RF differentiation of IgG3 and IgG4. By making site-directed mutations in the wild-type IgG3 or IgG4 human gamma constant genes, we showed that His-435 is an essential residue in RF binding to IgG for most WMac RFs. The allotypic polymorphism in IgG3 at 436 is not responsible for differences in previous reports of high-frequency IgG3 binding by WMac RFs. A amino acid loop in the CH2 domain of IgG4 proximal to the CH2-CH3 interface is important in WMac RF binding to IgG; a more distal CH2 loop in CH2 has a more variable effect on WMac RF binding. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating the glycosylation signal Asn-297 to another amino acid. Of all four IgG subclasses, only aglycosylated IgG3 was a better RF binding substrate than its glycosylated subclass counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo- fragment of polymerase I

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo⁻ fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.     

The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure

Bacterial class I release factors (RFs) are seen by cryo-electron microscopy (cryo-EM) to span the distance between the ribosomal decoding and peptidyl transferase centers during translation termination. The compact conformation of bacterial RF1 and RF2 observed in crystal structures will not span this distance, and large structural rearrangements of RFs have been suggested to play an important role in termination. We have collected small-angle X-ray scattering (SAXS) data from E. coli RF1 and from a functionally active truncated RF1 derivative. Theoretical scattering curves, calculated from crystal and cryo-EM structures, were compared with the experimental data, and extensive analyses of alternative conformations were made. Low-resolution models were constructed ab initio, and by rigid-body refinement using RF1 domains. The SAXS data were compatible with the open cryo-EM conformation of ribosome bound RFs and incompatible with the crystal conformation. These conclusions obviate the need for assuming large conformational changes in RFs during termination.     

Replication of semliki forest virus: polyadenylate in plus-strand RNA and polyuridylate in minus-strand RNA.

The 42S RNA from Semliki Forest virus contains a polyadenylate [poly(A)] sequence that is 80 to 90 residues long and is the 3'-terminus of the virion RNA. A poly(A) sequence of the same length was found in the plus strand of the replicative forms (RFs) and replicative intermediates (RIs) isolated 2 h after infection. In addition, both RFs and RIs contained a polyuridylate [poly(U)] sequence. No poly(U) was found in virion RNA, and thus the poly(U) sequence is in minus-strand RNA. The poly(U) from RFs was on the average 60 residues long, whereas that isolated from the RIs was 80 residues long. Poly(U) sequences isolated from RFs and RIs by digestion with RNase T1 contained 5'-phosphorylated pUp and ppUp residues, indicating that the poly(U) sequence was the 5'-terminus of the minus-strand RNA. The poly(U) sequence in RFs or RIs was free to bind to poly(A)-Sepharose only after denaturation of the RNAs, indicating that the poly(U) was hydrogen bonded to the poly(A) at the 3'-terminus of the plus-strand RNA in these molecules. When treated with 0.02 mug of RNase A per ml, both RFs and RIs yielded the same distribution of the three cores, RFI, RFII, and RFIII. The minus-strand RNA of both RFI and RFIII contained a poly(U) sequence. That from RFII did not. It is known that RFI is the double-stranded form of the 42S plus-strand RNA and that RFIII is the experimetnally derived double-stranded form of 26S mRNA. The poly(A) sequences in each are most likely transcribed directly from the poly(U) at the 5'-end of the 42S minus-strand RNA. The 26S mRNA thus represents the nucleotide sequence in that one-third of the 42S plus-strand RNA that includes its 3'-terminus.     

Formation and functional significance of the dark receptive fields of the cat visual cortex

Receptive fields (RFs) of single units in the 17th field of the visual cortex of immobilized cat were investigated under dark adaptation. The mean RF size was equal to 67 degrees and varied from 3 degrees up to 120 degrees. The RFs with centres located near gaze were from 3 degrees up to 120 degrees in dia, but with growth of excentricity the number of small RFs decreased, and in the region of 70 to 100 degrees from gaze only RFs with diameters equal to 100 degrees were found. The shape of "dark" RFs was either ellipsoidal (in most cases) or round. Detector properties (orientational, directional, size and velocity selectivity) of the "dark" RFs were significantly less manifest or absent. Under photopic light adaptation the same units reorganized their RFs to well known sizes and configuration. The hypothesis is discussed of the formation of local detector RF in the visual cortex in light adaptation by selective cortical inhibition which is activated in darkness only slightly. This view is an alternative to the commonly-accepted scheme of local cortical RF formation by the hierarchical and selective excitatory convergence.     

Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.     

Poly(C) sequence is located near the 5'-end of encephalomyocarditis virus RNA.

The distance between the poly(A) and poly(C) tracts in the molecules of encephalomyocarditis virus RNA has been estimated by two methods. The results indicate that these tracts are situated on the opposite ends of the viral RNA molecule. Evidence is presented that the poly(A) sequence in this molecule is located at the 3′-end. It is concluded that the poly(C) tract is situated at, or near, the 5′-end of the molecule.     

McrI: a novel class-II restriction endonuclease from Micrococcus cryophilus recognizing 5'-CGRY/CG-3'

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence. [sequence: see text] The novel enzyme in the presence of Mg2(+)-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages theta X174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.     

Life without A tail: New formats of long noncoding RNAs

While most long noncoding RNAs (lncRNAs) appear indistinguishable from mRNAs, having 5′ cap structures and 3′ poly(A) tails, recent work has revealed new formats. Rather than taking advantage of the canonical cleavage and polyadenylation for their 3′ end maturation, such lncRNAs are processed and stablized by a number of other mechanisms, including the RNase P cleavage to generate a mature 3′ end, or capped by snoRNP complexes at both ends, or by forming circular structures. Importantly, such lncRNAs have also been implicated in gene expression regulation in mammalian cells. Here, we highlight recent progress in our understanding of the biogenesis and function of lncRNAs without a poly(A) tail.This paper is part of a directed issue entitled: The Non-coding RNA Revolution.     

A cloned unique gene of drosophila melanogaster contains a repetitive 3′ exon whose sequence is present at the 3′ ends of many different mRNAs

We have started to study a cloned genomic DNA fragment ～7 kb long (denoted as H55) from the 7B3-4 region in the X chromosome of Drosophila melanogaster. The major part of the fragment is a single-copy sequence. It directs the synthesis of mRNA that makes up ～0.1% of the cytoplasmic poly(A)⁺ RNA from Drosophila embryos. The H55 gene is split by an intervening sequence, yielding a large single-copy exon and a small repetitive 3′ exon represented by hundreds of copies in the genome. This repetitive sequence (“suffix”) is also present at the 3′ ends of ～2% of all cytoplasmic poly(A)⁺ RNA chains.     

The 3'-terminal nucleotide sequences of lambda DNA

The base sequences of the 3′-termini of coliphage λ DNA have been analyzed by a new technique. Escherichia coli DNA polymerase I was used to add a single radioactive nucleotide to the 3′-OH terminus of one of the DNA strands. The DNA was then digested with pancreatic DNase I, and the resulting oligonucleotides were separated by two dimensional ionophoresis. Terminal oligonucleotides were identified by the presence of the radioactive label, and the base sequence of the labelled terminus was deduced from the base compositions of the terminal di-, tri-, tetra-, etc., oligonucleotides. It is found that the left 3′-terminus of λ DNA ends with the sequence d(pCpGpCpG) and the right 3′-terminus ends with the sequence d(pCpG).     

Possibilities of Triplex-Formation by 21-Mer Oligodeoxyribonucleotides as Analogs of Tetrahymena Pre-rRNA Fragments

Abstract

A potential DNA triple helix of 21-mer oligodeoxyribonucleotides was synthesized and characterized. The strands were chosen to study the interaction of internal guide and intervening sequences analogs as well as adjacent 3′and 5′exon parts around the splicing site of Tetrahymena pre-rRNA. Further in parallel works a series of different RNA and DNA strands was synthesized and combined yielding a suitable order of stability. Here we want to show an isolated examination of a DNA-strand triple helix with defined sequences containing a central mismatched base arrangement and T-A-T bases at the ends.     

Precise nucleotide location of the 5' ends of RNA-primed nascent light strands of mouse mitochondrial DNA

The mouse mitochondrial DNA origin of light-strand replication has been defined as a 32-nucleotide region located among five transfer RNA genes in the genomic sequence. A distinctive feature of this origin is its potential to form a perfectly complementary stem and 11-nucleotide loop structure. Previous studies have demonstrated that the 5′ ends of nascent light strands map within this region and a major trinucleotide ribosubstitution site in closed circular mouse mitochondrial DNA has been mapped within the stem sequence.Direct analysis and precise localization of the 5′ ends of nascent light strands indicate that essentially all 5′ ends are ribonucleotides mapping in the originspecific dyadic structure. The major 5′ end identified is the rG at position 5187 in the genomic sequence. Priming of replication most likely occurs within the loop portion of the potential dyad and continues for 2 to 16 nucleotides with a sharply defined switch to deoxyribonucleotide synthesis. This functional transition point is identical in map position to the trinucleotide ribosubstitution site in mature, closed circular mitochondrial DNA.     

STRUCTURAL CHARACTERIZATION OF THE TERMINAL DOMAINS OF LINEAR PLASMID‐LIKE DNA FROM THE GREEN ALGA ERNODESMIS (CHLOROPHYTA)1

Terminal regions of linear plasmid‐like DNA molecules from chloroplasts of the green alga Ernodesmis verticillata (Kützing) Børgesen were cloned and structurally characterized. Phosphorylation experiments with polynucleotide kinase indicated the presence of a 5′‐phosphate, but the data did not reveal any protective protein(s) at the 5′ end. To characterize the 3′ end of these molecules, homopolymer‐ (poly(G)‐) tailed molecules were annealed to and cloned into a linearized vector that was poly(C) tailed with terminal deoxynucleotidyl transferase. Sequencing analyses verified the heterogeneous nature of the molecules. Two distinct clones displayed extensive terminal inverted repeats (TIRs) at the 3′ end (94 and 433 nt). Shorter TIRs (3–6 nt) were identified at the 3′ end of most clones, which may serve to protect the ends. In fact, exonuclease III and λ exonuclease digested the plasmid‐like DNAs only after heat denaturation, signifying that conformational changes due to such treatment potentially make the 3′ and 5′ ends (respectively) susceptible to degradation. Multiple tandem and direct repeats were evident near the 3′ ends. A consensus sequence of 18+ nt was discovered in nearly every clone opposite the poly(G) tail, suggesting that this sequence has structural and/or functional significance. Pair‐wise sequence comparisons and the presence of repeats indicated that these novel molecules may be highly recombinant.     

Molecular recognition and catalysis in translation termination complexes

When the ribosome machinery reaches a stop codon in the mRNA, protein synthesis stops, and nascent polypeptide release is catalysed by class-I release factors (RFs); class-II RFs then promote the release of class-I RFs. Cryo electron microscopy structures of termination complexes and crystal structures of isolated factors have provided insights into key concepts such as bridging of active sites on the ribosome, and conformational changes that regulate the termination process. Recent crystal structures of the four possible functional ribosome complexes that contain the class-I RFs and the three stop codons have uncovered the molecular mechanisms by which RF1/RF2 (i) both recognise UAA, but discriminate specifically between UAG and UGA, and (ii) catalyse peptide release. Moreover, ongoing research also promises to reveal the structure-function relations of class-II RFs.