Straight core structure of DNA replication origins in the mammalian AMPD2 locus

Abstract

Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (~300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.

Ability of HMGB1 protein to bind to intrinsically bent and non-bent DNA sites in the AMPD2 gene amplicon

“…Electrophoretic behavior assays and in silico analyses was used by Lima Neto et al (2014) to map eleven IBDSs that are related to the replication origins oriGNA13, oriC, oriB, and oriA in the amplified domain of the AMPD2 gene of the Chinese hamster. In this study, we mapped the INBDSs present in this region.…”

“…The recombinant DNA was obtained from bacteria cultured for 16 h at 37°C in Luria-Bertani liquid medium by the cetyltrimethylammonium bromide (CTAB) extraction method and/or by the alkaline lysis method (Sambrook and Russell, 2001). Lima Neto et al (2014) mapped IBDSs from the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the amplified mammalian AMPD2 gene domain. The non-bent DNA fragments (Table 1) were selected by in silico analysis of replication initiation sites and flanking sequences from the amplified AMPD2 gene.…”

“…DNA fragments were obtained by PCR amplification, as described above. The bent DNA fragments and the primers sequences used for DNA amplification were as described by Lima Neto et al (2014). DNA fragments were purified by the ethanol/NaCl precipitation method (Sambrook and Russell, 2001) and quantified using the NanoDrop 2000c (Thermo Scientific).…”

“…In eukaryotes, IBDS have also been associated with replication initiation origin sites (Fiorini et al, 2001(Fiorini et al, , 2006aLima Neto et al, 2014). Moreover, there are indications that these structures are relevant to transcription (Fiorini et al, 2001), nucleosome condensation (Virstedt et al, 2004), recombination events (Milot et al, 1992), fragile genomic sites (Palin et al, 1998), nuclear matrix association sites (MARs), and MARs associated to replication origins (Anderson 1986, Fiorini et al, 2006b.…”

“…Characterizations of these regions in IBDS have previously been performed (Lima Neto et al, 2014). We also analyzed binding efficiency of HMGB1 protein to bent and non-bent DNA fragments mapped in the AMPD2 gene (oriA, oriB e oriC, oriGNAI3).…”