Analysis of Complex DNA Rearrangements during Early Stages of HAC Formation

Abstract

Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for 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.

Efficient formation of single-copy human artificial chromosomes

“…4). This is important because earlier generations of HACs typically formed in a manner accompanied by large-scale DNA sequence multimerization and even acquisition of portions (>100 kb) of host cell chromosomal DNA ( 16 , 37 , 38 ). YAC- Mm -4q21 LacO contains a single Fse I site (fig.…”

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

“…Using transformation-associated 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 TAR-assembled 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 – 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.…”

Concatenation of Transgenic DNA: Random or Orchestrated?

“…In nature, DNA concatenation could be used by viruses to amplify genome copy numbers [ 1 , 2 ]. Alternative applications of this principle include mitochondrial DNA replication [ 3 ], telomere maintenance [ 4 ], and multimerization of human artificial chromosomes [ 5 ] or replicative plasmids [ 6 ]. We will use the term “concatenation” in the context of transgene delivery, when tandemly arrayed transgene copies co-integrated into a genomic site.…”

“…One model predicts that a concatemer is built from one or more circular copies through de novo amplification by a rolling circle amplification or other means. This model could be inferred from the high efficiency of concatenation, the notion of occasionally observed identical transgene-transgene junctions [ 31 ], and the fact that cells possess mechanisms for gene amplification [ 5 , 32 , 33 ]. However, the amplification hypothesis was disproved by the studies with concatenation reporters, which demonstrated that copies in the concatemers are unique ( Figure 1 B–D) (see below).…”