Changes in gene copy number contribute to genomic instability, the onset and progression of cancer, developmental abnormalities, and adaptive potential. The origins of gene amplifications have remained elusive; however, one model suggests DNA rereplication could be a source of gene amplifications. The inability to determine which sequences are rereplicated and under what conditions have made it difficult to determine the validity of these models. Here we present RerepSeq, a technique that selectively fragments and enriches rereplicated DNA in preparation for next generation sequencing. RerepSeq is applicable to any species and can be utilized with low amounts of input DNA with rapid results. We validated RerepSeq by simulating DNA rereplication in yeast and human cells. Using RerepSeq, we demonstrate that rereplication induced in S. Cerevisiae by deregulated origin licensing is non-random and defined by broad domains that span multiple replication origins and topological boundaries.
Overall design
Rerep-seq leverages the semiconservative nature of DNA replication to selectively fragment and enrich rereplicated DNA. Cells are labeled with the thymidine analog BrdU for one cell cycle to allow BrdU incorporation into the newly replicated DNA strand, rereplicated regions incorporate BrdU in both strands. Genomic DNA from cells labeled with BrdU is purified and subjected to biochemical processing to induce ssDNA breaks at the sites of BrdU incorporation. This is performed by subjecting DNA to UVA treatment in the presence of Hoechst 33258 to photolyze the bromine from BrdU leaving deoxyuracil. Deoxyuracil is then removed by treatment with UDG (uracil DNA glycosylase) leaving an abasic site. The abasic site is then converted to a single strand DNA break by deoxyribose excision by APE1 (apurinic/apyrimidinic endodeoxyribonuclease). This results in nicked genomic DNA for normally replicated DNA, but staggered nicks producing dsDNA breaks in rereplicated DNA. The fragmented rereplicated DNA can then be isolated by size fractionation and analyzed by quantitative PCR or next-generation sequencing.
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