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Significance Extrachromosomal circular DNA (eccDNA) plays a role in human diseases such as cancer, but little is known about the impact of eccDNA in healthy human biology. Since eccDNA is a tiny fraction of nuclear DNA, artificial amplification has been employed to increase eccDNA amounts, resulting in the loss of native compositions. We developed an approach to enrich eccDNA populations at the native state (naïve small circular DNA, nscDNA) and investigated their origins in the human genome. We found that, in human sperm, the vast majority of nscDNA came from high-copy genomic regions, including the most variable regions between individuals. Because eccDNA can be incorporated back into chromosomes, eccDNA may promote human genetic variation., Extrachromosomal circular DNA (eccDNA) originates from linear chromosomal DNA in various human tissues under physiological and disease conditions. The genomic origins of eccDNA have largely been investigated using in vitro–amplified DNA. However, in vitro amplification obscures quantitative information by skewing the total population stoichiometry. In addition, the analyses have focused on eccDNA stemming from single-copy genomic regions, leaving eccDNA from multicopy regions unexamined. To address these issues, we isolated eccDNA without in vitro amplification (naïve small circular DNA, nscDNA) and assessed the populations quantitatively by integrated genomic, molecular, and cytogenetic approaches. nscDNA of up to tens of kilobases were successfully enriched by our approach and were predominantly derived from multicopy genomic regions including segmental duplications (SDs). SDs, which account for 5% of the human genome and are hotspots for copy number variations, were significantly overrepresented in sperm nscDNA, with three times more sequencing reads derived from SDs than from the entire single-copy regions. SDs were also overrepresented in mouse sperm nscDNA, which we estimated to comprise 0.2% of nuclear DNA. Considering that eccDNA can be integrated into chromosomes, germline-derived nscDNA may be a mediator of genome diversity.

The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12–21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.

Summary Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη) were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

Cancer continues to have a significant medical, economic, and psychological burden on patients worldwide. One strategy for reducing this impact involves the early detection of cancer biomarkers. Structural chromosomal aberrations called DNA palindromes have immense potential to be used as early markers in tumorigenesis. Palindromic sequences in the genome are structural genetic phenomena where a sequence of nucleotides is identical to its reverse compliment. Various independent studies have identified this distinctive pattern of genome instability in a wide range of cancers including breast, pancreatic, and ovarian cancers. We have shown previously that DNA palindromes underlie the amplification of ERBB2 in breast cancer. This suggests that DNA palindromes are the candidate breast cancer biomarker. A convenient and sensitive approach for identifying palindromes in breast tumor DNA could be a promising diagnostic test. Palindrome detection from tumor DNA in blood (liquid biopsy) could circumvent the need for invasive biopsies. Here, we develop an unbiased genome-wide technique for detecting DNA palindromes as a genomic target for breast cancer screening in the clinic. Using genome-wide analysis of palindrome formation (GAPF), we are able to enrich for DNA palindromes from samples of genomic DNA isolated from cells and tumor tissues. Following a denaturation step, DNA palindromes naturally form double-stranded DNA with itself because these sequences are physically tethered together and can form energetically favorable base pairs. Non-palindromic DNA remains single-stranded and subsequently removed by single-strand specific nucleases thereby dramatically improving the signal-to-noise ratio. This approach confers advantages over other methods because it simultaneously (1) amplifies target signal and (2) reduces background noise without employing sub-sampling techniques. We first used a cancer cell line Colo320DM containing a known palindromic junction and normal fibroblasts IMR90. By integrating GAPF with next generation sequencing (GAPF-Seq), we mapped sequenced reads to a reference genome and developed an algorithm to demarcate DNA palindromes throughout the genome. We are able to identify the known DNA palindrome in Colo320DM in a 10-fold dilution in IMR90 DNA. We then used GAPF-Seq to identify tumor-specific DNA palindromes in matched pairs of tumor and normal breast cancer tissue. From 100 ng of input DNA, we generated a profile of more than 300 tumor-specific DNA palindromes that were not found in the matched normal tissue. To optimize GAPF for use in cancer screening, we expect to detect DNA palindromes in the presence of 100-fold excess of non-palindromic DNA from as little as 10 ng of input DNA. We will then use matched plasma and buffy coat samples to generate whole-genome palindromic profiles from liquid biopsies. The data can also be used for tracing clonal evolution by comparing individual bins in the palindrome profiles of tumor tissue and plasma cell-free DNA from the same patient. Finally, we will optimize GAPF-Seq for minimal computational processing and data storage requirements. Due to the amplification of palindromes and reduction of background signal, GAPF-Seq may be able to identify tumor-specific breast cancer markers from as few as 1 million reads and 250 MB of data, which would be ideal for wide use in screening for breast tumors in the general population and monitoring disease outcomes with multiple blood samplings. We believe GAPF-Seq addresses a need to develop an unbiased, genome-wide, and rapid approach for breast cancer screening. Citation Format: Michael M. Murata, Lila Mouakkad, Dolores Di Vizio, Armando E. Giuliano, Hisashi Tanaka. DNA palindromes as novel genomic targets for unbiased, genome-wide, and rapid pan-cancer screening [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-24.