Most relevant achievements

     In our group we have been studying the topology and replication of DNA in different prokaryotic and eukaryotic systems for the last 20 years. We were the first to characterize unidirectional DNA replication by means of two-dimensional agarose gel electrophoresis (Martín-Parras et al., 1991). We were the first to report that in those plasmids and minichromosomes containing more than one potential origin, initiation of DNA replication occurs at only one of the potential origins per replication round, regardless of origin orientation (Schvartzman et al., 1990; Martín-Parras et al., 1992; Viguera et al., 1996). This phenomenon is called "origin interference" and its nature is still unknown. In those bacterial plasmids with inversely oriented unidirectional origins, the silent origin acts as a polar barrier for the replication fork initiated at the other origin (Viguera et al., 1996). This is due to the incompetence of Escherichia coli DnaB helicase, which leads the replication fork in vivo, to unwind the RNA 3' end of RNA-DNA hybrids (Santamaría et al., 1998). Blockage of the replication fork leads to the accumulation of replication intermediates (RIs) containing an internal bubble, which may serve as substrate for inadvertent knotting events resulting from topo II-type action that tend to fix some of the crossings present in interwound supercoiled molecules (Olavarrieta et al., 2002a,b,c; Viguera et al., 1996). For this reason most of these nodes have a positive sign (Sogo et al., 1999). Blockage of the replication fork is not just a pause but permanent, as it leads to a premature termination event (Santamaría et al., 2000). Interestingly, RIs containing an internal bubble may undergo reversion of the fork, at least in vitro (Viguera et al., 2000). We were also among the first to characterize positive-supercoiling induced replication-fork reversal (Olavarrieta et al., 2002; Viguera et al., 2000; Fierro-Fernández et al., 2007a,b). In eukaryotic plasmids with a bidirectional origin, termination does not occur at a fixed site but may take place anywhere depending on the firing synchrony of both forks at the origin and the rate of forks progression (Santamaría et al., 2000). We were also the first to use two-dimensional agarose gel electrophoresis and electron microscopy to identify all the different topological forms that autonomously replicating circular plasmids and minichromosomes can adopt in living cells (Martín-Parras et al., 1998; Mayán-Santos et al., 2007; Schvartzman et al., 2010). Our group participated also in a joint effort to find that the genome of Streptococcus pneumoniae is organized in topology-reacting gene clusters (Ferrándiz et al., 2010). We also found that in Saccharomyces cerevisiae, those circular minichromosomes containing the rDNA replication fork barrier (RFB) are unstable in ?sir2 strains and integrate at the rDNA locus by homologous recombination (Benguría et al., 2003). Curiously, integration occurs only at two specific sites: the RFB and the 3’ end of the 35S precursor gene (Mayán-Santos et al., 2008). We were the first to characterize the interplay of supercoiling and decatenation during the segregation of sister duplexes in bacterial plasmids (Martínez-Robles et al., 2009). Finally, we demonstrated that positive supercoiling drives decatenation by topoisomerase II in eukaryotes (Baxter et al., 2011).

Identification of Knotted Replication Bubbles

Positive supercoiling leads to replication fork reversal