In Vitro Murine Leukemia Retroviral Integration and Structure Fluctuation of Target DNA

Abstract

Integration of the retroviral genome into host DNA is a critical step in the life cycle of a retrovirus. Although assays for in vitro integration have been developed, the actual DNA sequences targeted by murine leukemia retrovirus (MLV) during in vitro reproduction are unknown. While previous studies used artificial target sequences, we developed an assay using target DNA sequences from common MLV integration sites in Stat5a and c-myc in the genome of murine lymphomas and successfully integrated MLV into the target DNA in vitro. We calculated the free energy change during folding of the target sequence DNA and found a close correlation between the calculated free energy change and the number of integrations. Indeed, the integrations closely correlated with fluctuation of the structure of the target DNA segment. These data suggest that the fluctuation may generate a DNA structure favorable for in vitro integration into the target DNA. The approach described here can provide data on the biochemical properties of the integration reaction to which the target DNA structure may contribute.

In Vitro HIV-1 LTR Integration into T-Cell Activation Gene CD27 Segment and the Decoy Effect of Modified-Sequence DNA

“…In the current study, we modified the protocol further by omitting the PCR reaction and using the actual human CD27 gene as the target DNA sequence (Fig. 1B) [7], [11].…”

“…The details of target sequence and the replacement nucleotides were represented in text. The percentage of integration was estimated by determining the copy number of the products of integration into the target DNA compared to the copy number of the products of integration into the whole-sequence DNA (i.e., the target DNA plus the plasmid), according to previously reported methods [7], [9], [11]. First, the number of E. coli colonies exhibiting integration into the target segment was determined.…”

High-Resolution Breakpoint Analysis Provides Evidence for the Sequence-Directed Nature of Genome Rearrangements in Hereditary Disorders

“…They often coincide with other, comparably simpler structures, such as cruciform, hairpins, H and Z-DNA, all of which we found abundantly at the hGR and nhGR BPs. The preponderance of nhGR BPs to adopt cruciform structures is not surprising in the light that they are preferred targets for retroviral integration into host genomes via nonhomologous recombination [Katz et al, 1998;Tsuruyama et al, 2012], and have been implicated as the likely source for single and double strand breaks in the genome [Bacolla et al, 2004]. Finally, the value and usefulness of novel data and hypotheses often derive from the accuracy of their predictions.…”

Hotspots of MLV integration in the hematopoietic tumor genome

“…It has been suggested that the terminal ends of the dsDNA might be critical for integration, and that the presence of a dinucleotide capsid at the terminal 3′-ends is particularly essential. 13 , 41 Subsequently, the free energy change released from the broken phosphodiester bonds in the host dsDNA promotes the formation of new bonds joining the viral DNA ends to the ends of cleaved host DNA. DNA synthesis extends from the host DNA flanking the host-viral DNA junctions and fills in the gaps adjacent to the viral DNA, displacing the viral DNA ends.…”

“… 9 , 10 DNA secondary structural fluctuation around an integration hotspot might contribute to the open chromatin structure in the transcriptionally active site that would likely promote proviral integration after the physical breakage of the double-stranded DNA (dsDNA). 13 , 14 …”