Noncanonical DNA Elements in the Lamin B2 Origin of DNA Replication

Abstract

DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT) n (dA) n sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/ GGAAAAAAAAAAG (3900 -3912) and CCTTTTTTTTC/ GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mM Tris base, 89 mM boric acid, and 2 mM MgCl 2 ) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT) 40 do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads.Eukaryotic origins of DNA replication are sequences derived from fixed chromosomal regions that accommodate one or more DNA replication start sites and direct binding of origin recognition complexes (ORCs).1 These regions are typically intergenic, adjacent to transcriptionally active genes, and vary in size from several hundred to several thousand base pairs, which is a consequence of natural divergence and/or of different mapping procedures (1-3). In some cases, origins colocalize with matrix attachment sites (4) or require two functional elements, only one of which serves for initiation, whereas both bind ORC (5-7). In addition, DNA replication origins do not share a common consensus sequence but still act as replicators when transferred to another chromosomal location (2,8,9). This important ability demonstrates that elements contained in naked DNA build structural basis for initiation of DNA synthesis.Apart from AT richness, which seems to be the general feature of eukaryotic origins of DNA replication, elements that account for common function are very poorly defined (3, 10, 11). They could include (dT) n (dA) n sequences predominantly distributed in a mirror repeat manner, and as reported, involved in initiator protein binding (5, 12-18). The structural impact of these sequences was not investigated, but numerous other studies confirm that A and T tracts (depending on their distribution and size) could profoundly affect geometry of grooves, bending, and the ability to bend or formation of loops and multistranded structures (19). Ha...

Functional interactions of DNA topoisomerases with a human replication origin

“…The replacement of a 10 T stretch, shown to spontaneously acquire in vitro unusual, probably triple‐stranded, structures (Kusic et al , 2005) and located close to the in vivo topo I site on the upper strand, with a grossly modified sequence, abolished the two nearby in vitro cleavage sites but not the far away ones (see Figure 2B, lanes 4 and 13, and C). The interaction with the lower strand was abrogated instead by replacement of six nucleotides comprising the topo I binding site (see Figure 2B, lane 7, and C).…”

Homeotic proteins participate in the function of human-DNA replication origins

“…DNA topology and chromatin structure are closely interrelated, affect each other and depend also on relatively long-range effects of DNA sequence, such as the tendency to form unusual DNA structures (triple strands, Hoogsteen structures) and to bend the duplex. The lamin B2 origin sequence is characterized by an intrinsic tendency to form such unusual structures (47) and contains a bent DNA sequence between nt 3923 and 3928. Furthermore, topological status is clearly critical for origin recognition by ORC in yeast and Drosophila origins (48,49), while topo I and II bind precise sites in the lamin B2 pre-RC area in precise moments of the cell cycle, topo I being essential for synthesis initiation (44,50).…”

HsOrc4-dependent DNA remodeling of the ori-β DHFR replicator

“…5B). A similar model was suggested in previous studies, based on experiments on linear fragments or on plasmid studies [26,27]. Since the AT region melts easily and is capable of forming non-canonical structures, the triple helix is not so unexpected.…”