Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Abstract

<jats:title>Abstract</jats:title><jats:p>Human Artificial Chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN) and possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.</jats:p>

Transformation-associated recombination (TAR) cloning and its applications for gene function; genome architecture and evolution; biotechnology and biomedicine

“…Using transformationassociated recombination in yeast, it was shown that 2mer or 4mer or 5mer alphoid DNA repeats consisting of alphoid 170 bp monomers and having the ends homologous to each other may be one-step TARassembled into long synthetic alphoid DNA arrays varying in size from 50 to 140 kb (Step 1: Figure 11). After transfection of such arrays into human cells, de novo HACs are generated, ranging in size from 1 to 10 Mb due to amplification of the input alphoid DNA arrays (Steps 2 and 3) [139][140][141][142][143]. Because any nucleotide in the original dimer can be easily changed before its amplification, this TAR-based method allows to identify the critical regions of the alphoid repeat for de novo centromere seeding.…”