Nava Baran

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the Km for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.

Unusual sequence element found at the end of an amplicon.

In a polyomavirus-transformed rat cell line, designated LPT, the polyomavirus DNA is integrated into a single chromosomal site. Treatment of LPT cells with carcinogens induces amplification of the integrated virus DNA and flanking cellular sequences. We show that the amplification is arrested within a specific cell DNA segment that maps 1.3 to 1.85 kilobases beyond one virus-cell DNA junction, defined as the left junction. We also present the sequence of an 897-base-pair fragment spanning the arrest site. This fragment contains an unusual sequence element, which consists of two contiguous components, a potential cruciform with stems of 6 base pairs and a d(G-A)27 X d(T-C)27 tract, and maps 1,497 to 1,564 nucleotides beyond the left junction. The possibility that this unusual sequence plays a role in the arrest of the amplification process is discussed.

In situ hybridization analysis of polyoma DNA replication in an inducible line of polyoma transformed cells

Abstract In situ hybridization has been used to study polyoma DNA replication in a clonal derivative of the inducible LPT line of polyoma-transformed cells designated as clone 1A. This study has shown that in clone 1A cultures maintained under normal growth conditions, 4–25 in 10,000 cells are spontaneously induced to synthesize polyoma DNA at an enhanced rate. In cultures exposed to mitomycin C (MMC), the percentage of induced cells remains approximately equal to the spontaneous level for 9 hr, and then increases for at least 24 hr up to 30–57% as more and more cells are asynchronously recruited to replicate the virus DNA. DNA reassociation kinetics and in situ hybridization have been used to determine the amount and distribution of polyoma DNA accumulated within clone 1A cells. These measurements have shown that a single induced cell in an MMCtreated culture produces 24,500 genome-equivalents of the virus DNA; second, that the average yield of virus DNA in a normally growing culture is only 41.7 genome-equivalents per cell; however, a single spontaneously induced cell in this culture produces as much virus DNA as an MMC-induced cell; third, that all the virus DNA molecules are found within the nuclei and many are clustered in aggregates containing up to 2000 genome-equivalents. We discuss the implications of these findings regarding the regulation of polyoma DNA replication in the LPT line.

Interference Footprinting Analysis of Telomerase Elongation Complexes

ABSTRACT Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymenatelomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single 32P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3′-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3′-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3′ ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.

Integration of polyoma virus DNA into chromosomal DNA in transformed rat cells causes deletion of flanking cell sequences.

In order to find out whether polyoma virus (Py) integration into chromosomes causes rearrangements in the cell DNA flanking the integration site, we have mapped the flanking sequences in the inducible LPT line of Py-transformed rat cells and the corresponding sequences in normal rat fibroblasts, and then compared the two maps. To carry out this study we have cloned a segment including Py DNA and flanking sequences in the bacteriophage vector lambda gt WES and subcloned the flanking cell DNA in a bacterial plasmid. We performed a Southern blot analysis of LPT and rat fibroblast DNA digested with various restriction enzymes and used the cloned flanking cell DNA and Py DNA as hybridization probes. Autoradiography of the LPT DNA blots revealed two sets of fragments. One set includes fragments containing both Py and cell DNA sequences; the second set consists of fragments which contain no virus DNA sequences, and are identical to the fragments observed in the corresponding normal rat DNA digests. These data indicate that LPT cells are heterozygous with respect to the Py inserts. The same data were used to map the flanking sequences in the two types of cells. A comparison between the two maps revealed that a 3.0 kb cell DNA segment, which is located next to the unoccupied integration site in the normal rat chromosomes, has been deleted from the LPT chromosome which carries Py DNA, but not from the LPT chromosome which does not carry the virus DNA. The implications for papovavirus integration are discussed.

Quantification of diverse virus populations in the environment using the polony method

Viruses are globally abundant and extremely diverse in their genetic make-up and in the hosts they infect. Although they influence the abundance, diversity and evolution of their hosts, current methods are inadequate for gaining a quantitative understanding of their impact on these processes. Here we report the adaptation of the solid-phase single-molecule PCR polony method for the quantification of taxonomically relevant groups of diverse viruses. Using T7-like cyanophages as our model, we found the polony method to be far superior to regular quantitative PCR methods and droplet digital PCR when degenerate primers were used to encompass the group’s diversity. This method revealed that T7-like cyanophages were highly abundant in the Red Sea in spring 2013, reaching 770,000 phages ml−1, and displaying a similar depth distribution pattern to cyanobacteria. Furthermore, the abundances of two major clades within the T7-like cyanophages differed dramatically throughout the water column: clade B phages that carry the psbA photosynthesis gene and infect either Synechococcus or Prochlorococcus were at least 20-fold more abundant than clade A phages that lack psbA and infect Synechococcus hosts. Such measurements are of paramount importance for understanding virus population dynamics and the impact of viruses on different microbial taxa and for modelling viral influence on ecosystem functioning on a global scale.Adaptation of the polony method allows numerical abundances of diverse viral groups to be quantified in environmental samples, and reveals that clade B T7-like cyanophages that carry the psbA gene are more abundant in the Red Sea than clade A phages.

Organization and Replication of Chromosome-Associated Polyoma Virus DNA and Flanking Cellular Sequences in Polyoma-Transformed Rat Cells

In this chapter we review studies on the organization and replication of polyoma virus (Py) DNA sequences which have integrated into chromosomal DNA, and of cellular DNA flanking the Py integration site, in an inducible line of polyoma-transformed rat cells designated the LPT line. We also discuss the relationship between replication of the chromosome-associated viral DNA and excision of free viral DNA molecules in LPT cells treated with virus-inducing agents. These studies have shown that LPT cells contain multiple copies of Py DNA integrated into a single chromosomal site. These copies are not evenly distributed among the cells. Instead, various Py insertions include 1, 2, 3,…whole viral genomes arranged in a direct tandem repeat. A 3.0 kilobase pairs (kb) deletion of cellular DNA was discovered next to the Py integration site. LPT cells were found to be heterozygous with respect to the viral insertions and the cellular deletion. In LPT cultures treated with mitomycin c, the most effective inducing agent in this system, replication of Py DNA is enhanced after lag periods which vary in individual cells between 9 and 20 hours. DNA synthesis is repeatedly initiated within the viral insertions and the replication forks proceed in opposite directions. The leftward moving forks were analyzed in detail and found to cross the left viral DNA-cell DNA boundary and to proceed into the flanking cellular DNA sequences. Fork movement is halted within a 0.30 kb segment which maps about 2.0 kb away from the boundary. This segment may include a termination site of a normal cellular replicon. Similar results were obtained in LPT cells exposed to bromodeoxyuridine and ultraviolet light. Extrachromosomal circular Py DNA monomers are excised and replicated in induced LPT cells after the lag phase. The viral DNA monomers are encapsidated and converted into infectious virions. Excision of free viral DNA molecules and virus synthesis do not occur in an LPT subclone which contains chromosome-associated tandemly repeated viral DNA, but in which replication of the viral DNA cannot be induced. Models of papovavirus integration and induction based on studies of the LPT line and of other Py and SV40 transformants are discussed.