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.
Highlights
Bind origin recognition complexes (ORCs) [5,6,7]
Experiments were performed with origin fragment lbo I-(3813– 4052), previously shown to encompass the initiation site and regions interacting with ORC in vivo and in vitro
In this study we have probed the structure of the human lamin B2 origin, focusing on sequence elements common for eukaryotic DNA replication origins in general and using chemical and electrophoretic assays that we considered suitable for analysis of noncircular molecules that are several hundred base pairs long
Summary
Bind ORC [5,6,7]. 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]. 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] Having this in mind, we have probed the structure and shape of partly overlapping fragments lbo I-(3813– 4052) and lbo II(3871– 4187) isolated from the human lamin B2 origin of DNA replication. We have probed the structure and shape of partly overlapping fragments lbo I-(3813– 4052) and lbo II(3871– 4187) isolated from the human lamin B2 origin of DNA replication This origin is very well characterized, accommodates the defined start site of DNA synthesis, binds ORC in living cells and in vitro, and supports the initiation of DNA synthesis at ectopic chromosomal locations (2, 18, 20 – 23). Our combined results show that at neutral pH, low ionic strength, and in the presence of Mg2ϩ ions, linear fragments of the lamin B2 contain nonhydrogen bonded (dT)n (dA)n tracts that most likely make part of the short triple stranded structures
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