DNA Bending in the Replication Zone of the C3 DNA Puff Amplicon of Rhynchosciara americana (Diptera: Sciaridae)

Abstract

Intrinsic bent DNA sites were identified in the 4289 bp segment encompassing the replication zone which directs DNA amplification and transcription of the C3-22 gene of Rhynchosciara americana. Restriction fragments showed reduced electrophoretic mobility in polyacrylamide gels. The 2D modeling of the 3D DNA path and the ENDS ratio values obtained from the dinucleotide wedge model of Trifonov revealed the presence of four major bent sites, positioned at nucleotides -6753, -5433, -5133 and -4757. Sequence analysis showed that these bends are composed of 2-6 bp dA.dT tracts in phase with the DNA helical repeat. The circular permutation analysis permitted the verification that the fragments containing the bending sites promote curvature in other sequence contexts. Computer analyses of the 4289 bp sequence revealed low helical stability (DeltaG values), negative roll angles indicating a narrow minor groove and a putative matrix attachment region. The data presented in this paper add to information about the structural features involved in this amplified segment.

Sequence-Directed DNA Curvature in Replication Origins Segments

“…The generated recombinant pBendBlue plasmids are then introduced into host bacteria by means of DNA transformation and selected by a direct assay. The plasmid DNA is isolated by minipreparation and digested in independent reactions with suitable restriction enzymes, which recognize the duplicated pBendBlue polylinker, yielding a set of circularly permutated DNA fragments of identical size, but with a permutated bent position (Fiorini et al, 2006a;Gimenes et al, 2008b;Gimenes et al, 2009;Sperbeck & Wistow, 1998 Fig. 4.…”

“…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).…”

“…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).…”

“…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.…”

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

“…It is known that the intrinsic curvature of DNA plays an important role in cellular processes such as nucleosome compaction (8), recombination processes (9), fragile sites (10), tandem repeated sequences (11), replication origins (12,13), Scaffold/Matrix Attachment Regions (S/MARs) (14) and MARs associated with replication origins (13,15).…”