RSSsite: a reference database and prediction tool for the identification of cryptic Recombination Signal Sequences in human and murine genomes

Recombination signal sequences (RSSs) flanking V, D and J gene segments are recognized and cut by the VDJ recombinase during development of B and T lymphocytes. All RSSs are composed of seven conserved nucleotides, followed by a spacer (containing either 12 ± 1 or 23 ± 1 poorly conserved nucleotides) and a conserved nonamer. Errors in V(D)J recombination, including cleavage of cryptic RSS outside the immunoglobulin and T cell receptor loci, are associated with oncogenic translocations observed in some lymphoid malignancies. We present in this paper the RSSsite web server, which is available from the address http://www.itb.cnr.it/rss. RSSsite consists of a web-accessible database, RSSdb, for the identification of pre-computed potential RSSs, and of the related search tool, DnaGrab, which allows the scoring of potential RSSs in user-supplied sequences. This latter algorithm makes use of probability models, which can be recasted to Bayesian network, taking into account correlations between groups of positions of a sequence, developed starting from specific reference sets of RSSs. In validation laboratory experiments, we selected 33 predicted cryptic RSSs (cRSSs) from 11 chromosomal regions outside the immunoglobulin and TCR loci for functional testing

Transposon Excision from an Atypical Site: A Mechanism of Evolution of Novel Transposable Elements

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3′ to 5′ trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations

Terminal Deoxynucleotidyl Transferase: The Story of A Misguided DNA Polymerase

Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis

Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage.

Cleavage of V(D)J recombination signals by purified RAG1 and RAG2 proteins permits the dissection of DNA structure and sequence requirements. The two recognition elements of a signal (nonamer and heptamer) are used differently, and their cooperation depends on correct helical phasing. The nonamer is most important for initial binding, while efficient nicking and hairpin formation require the heptamer sequence. Both nicking and hairpin formation are remarkably tolerant of variations in DNA structure. Certain flanking sequences inhibit hairpin formation, but this can be bypassed by base unpairing, and even a completely single-stranded signal sequence is well utilized. We suggest that DNA unpairing around the signal-coding border is essential for the initiation of V(D)J combination