Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites

Abstract

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5)

Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation

“…How can a single nucleotide substitution T to C lead to the inactivation of a replication origin? The T/C SNP could prevent the binding and proper function of proteins that bend DNA (Mysiak et al, 2004a,b) or trigger DNA looping (Snyder et al, 1986; Zahn and Blattner, 1987; Hsieh et al, 1991; Balani et al, 2010). Less flexible DNA could be more difficult to access for proteins, such as replication factors.…”

Sequence-Directed DNA Curvature in Replication Origins Segments

“…In the same work, the in silico analysis of the replication origin segments from X. fastidiosa Temecula, Bacillus subtilis and Escherichia coli showed that all of the replication sites, with some variability in their helical parameters, displayed curved segments. In eukaryotes cells, our work was associated with gene amplified domains in developmental systems (Fiorini et al, 2001;2006a;Gimenes et al, 2009) and induced gene amplified segments in mammalian culture cells (Balani et al, 2010). Using the developmental amplified models, we analyzed amplicons from the gene BhC4-1 from Bradysia hygida (Fiorini et al, 2001), an amplified segment of the C3-22 gene from Rhynchosciara americana and in the segment DAFC-66D from chromosome 3 of Drosophila melanogaster, which contains the amplification control element ACE3 and the replication origin ori-β (Gimenes et al, 2009).…”

“…Nucleotide sequences of approximately 100 bp containing DNA bent sites are amplified from a DNA sample (approximately 50 ng/µl), and the primers used to amplify the DNA bent sites are designed with the restriction sites of XbaI and SalI at the ends for posterior cloning into a circular permutation vector such as pBendBlue (Sperbeck & Wistow, 1998). The PCR amplified fragments can be cloned into any PCR cloning vector such as the pGEM-T Easy Vector System (Promega), pMOSBLUE (GE Healthcare), pTZ57R/T (Balani et al, 2010;Gimenes et al, 2009;Hägg et al, 2004;Rodríguez-Lecompte et al, 2001) or the TOPO® PCR Cloning vector (Invitrogen). The recombinant plasmids may be sent for sequencing to confirm the identity of the insert.…”

“…The correlation between specific chromosomal regions and the topological features of DNA is an exciting area of research. Our laboratory has been devoted to mapping the presence of intrinsically bent DNA sites in regions of replication initiation in prokaryotes (Gimenes et al, 2008a), replication origins and promoter regions in gene amplified domains of eukaryotes (Balani et al, 2010;Fiorini et al, 2001Fiorini et al, , 2006aGimenes et al, 2009;Gouveia et al, 2008) and in recombination regions from the eukaryotes genome (Barbosa et al, 2008). Here we discuss the sequence and topological features of intrinsically bent DNA sites as well the methodology used to explore these sites in replication origin segments.…”

“…Analyses of replication intermediates using alternative methods indicated that the replication origin lacked a nucleotide consensus sequence (Biamonti et al, 2003;DePamphilis, 1999;Toledo et al, 1998;Tower, 2004). The activity of the initiation site for replication in metazoans, which lacks a consensus sequence, can be influenced by chromatin structure and/or selected by epigenetic factors (Anglana et al, 2003;Balani et al, 2010;Courbet et al, 2008;Debatisse et al, 2004;Fiorini et al, 2006a;Gimenes et al, 2009;Stehle et al, 2003). The presence of foci of replication (replication factories) that bring together numerous sites of replication initiation and the association of the nuclear matrix (S/MAR) demonstrates the importance of chromosome structure in DNA replication process (Anachkova et al, 2005;Courbet et al, 2008;Jackson, 2003;Newport & Yan, 1996).…”