Wei, X.; Gong, W.; Zheng, Y.; Zhang, J.; Wei, X.; Peng, C.; He, X.; Jiang, C.

Tandem repeats (TR) are common genomic elements with major functional consequences. Yet, the evolutionary trajectory for their formation remains poorly understood. One proposed mechanism is microhomology-mediated tandem duplication (MTD), in which single-copy DNA segments flanked by microhomology undergo tandem duplication (TD) and can further expand into TRs. Although MTD was first identified in the fission yeast Schizosaccharomyces pombe, its evolutionary conservation and postulated role in TR evolution had not been established. Using whole-genome deep sequencing and new analytical tools, we show that MTDs occur de novo universally across bacteria, archaea, fungi, and viruses. Further analysis of 2,245 reference genomes and millions of isolate genomes from 103 prokaryotic and eukaryotic microbial species, combined with human population TD data, somatic-germline mutations, and disease-associated variants, reveals that MTDs are consistently the dominant TD-forming mechanism across domains of life. Evidence suggests that MTDs have initiated the formation of most existing TRs in genomes. Importantly, MTDs also prevail in human pathogenic TR mutations, including those linked to cancers. Mechanistically, deletion of the conserved mutator gene Rad27 specifically increased MTD frequency in the budding yeast Saccharomyces cerevisiae, implicating Rad27-mediated Okazaki fragment maturation in MTD formation. These findings establish MTD as a universal and functionally significant mechanism for TR genesis.