Recombination hotspot activity of hypervariable minisatellite DNA requires minisatellite DNA binding proteins

Abstract

Hypervariable minisatellite DNA repeats are found at tens of thousands of loci in the mammalian genome. These sequences stimulate homologous recombination in mammalian cells [Cell 60:95-103]. To test the hypothesis that protein-DNA interaction is required for hotspot function in vivo, we determined whether a second protein binding nearby could abolish hotspot activity. Intermolecular recombination between pairs of plasmid substrates was measured in the presence or absence of the cis-acting recombination hotspot and in the presence or absence of the second trans-acting DNA binding protein. Minisatellite DNA had hotspot activity in two cell lines, but lacked hotspot activity in two closely related cell lines expressing a site-specific helicase that bound to DNA adjacent to the hotspot. Suppression of hotspot function occurred for both replicating and non-replicating recombination substrates. These results indicate that hotspot activity in vivo requires site occupancy by minisatellite DNA binding proteins.

Fig. 1

Homologous recombination substrates Circular plasmids are presented as linear schematics for the sake of clarity. (a) The recombination substrates were derived from pSV2neo which contains the neo gene (white box) that can be expressed in E. coli and in mammalian cells due to the presence of the SV40 virus early promoter and poly(A) addition sequences (black boxes). In addition, the plasmids can replicate as episomes in mammalian cells expressing SV40 virus large T antigen due to the presence of the SV40 origin of replication (56) Deletion left (DL) and deletion right (DR) were created by removing NarI-NarI and NaeI-NaeI restriction fragments, respectively (57). (b) Expanded detail of substrates. The hypervariable minisatellite (SAT) recombination hotspot was cloned in the position of the StuI-HindIII fragment in pSV2neo and the various deletion plasmids (49). This places the minisatellite DNA, and its binding proteins (Msbp) (53), 8 bp away from the T antigen binding site.

Fig. 2

Strategy to determine recombinant frequencies. The assay relies upon homologous recombination to generate a dominant, selectable marker gene (neo⁻) from two nonfunctional, nonreverting deletion genes that are cotransfected into the same cell. Both reciprocal (×) and nonreciprocal (not shown) recombination events can occur (48, 49). For cells incapable of supporting plasmid replication, the selectable gene integrates randomly into the genome and confers cellular resistance to G418. The number of G418 resistant colonies generated is directly proportional to the recombinant frequency. For cells permitting plasmid replication the episomal DNA is purified and transfected into recombination deficient E. coli, which reveals the recombinant frequency (neo⁺, kanamycin resistant) relative to the transformation efficiency (ampicillin resistant).

Fig. 3

Replication efficiencies of substrates. (a) Autoradiograph of replicated DNA isolated from COS-1 cells. Cells were cotransfected with the internal replication control plasmid p2X21ori and the various recombination substrates. After 56 h of incubation low molecular weight DNA was extracted, linearized by SalI restriction enzyme digestion, and the unreplicated input DNA was destroyed by a five-fold excess (relative to SalI) of DpnI restriction enzyme. Southern blotting revealed the relative amounts of replicated p2X21ori and the pSV2neo derivatives. The replication efficiency of each plasmid was determined by laser densitometry of the autoradiograph. (b) DNA sequence changes at the T antigen binding site and relative replication efficiencies of the recombination substrates The substrates pDLΔSH and pDLΔSS (59) have replication efficiencies comparable to those of pDLSAT and pDRSAT, permitting a direct comparison of recombinant frequencies between pairs of substrates with matched replication rates.

Fig. 4

Model of function. A normal, basal level of recombination occurs in cells lacking the minisatellite DNA hotspot and this basal frequency of recombination is essentially unaffected by T antigen binding or substrate replication. In the absence of T antigen, interaction of minisatellite DNA binding proteins (Msbp) (53) promotes recombination in nearby DNA intervals. Binding of T antigen displaces the minisatellite DNA binding proteins and renders the hotspot nonfunctional.