Unraveling the complexity of asymmetric DNA replication: Advancements in ribonucleotide mapping techniques and beyond

DNA replication is a fundamental process for cell proliferation, governed by intricate mechanisms involving leading and lagging strand synthesis. In eukaryotes, canonical DNA replication occurs during the S phase of the cell cycle, facilitated by various components of the replicative machinery at sites known as replication origins. Leading and lagging strands exhibit distinct replication dynamics, with leading strand replication being relatively straightforward compared to the complex synthesis of lagging strands involving Okazaki fragment maturation. Central to DNA synthesis are DNA polymerases, with Polα, Polε, and Polδ playing pivotal roles, each specializing in specific tasks during replication. Notably, leading and lagging strands are replicated by different polymerases, contributing to the division of labor in DNA replication. Understanding the enzymology of asymmetric DNA replication has been challenging, with methods relying on ribonucleotide incorporation and next-generation sequencing techniques offering comprehensive insights. These methodologies, such as HydEn-seq, PU-seq, ribose-seq, and emRiboSeq, offer insights into polymerase activity and strand synthesis, aiding in understanding DNA replication dynamics. Recent advancements include novel conditional mutants for ribonucleotide excision repair, enzymatic cleavage alternatives, and unified pipelines for data analysis. Further developments in adapting techniques to different organisms, studying non-canonical polymerases, and exploring new sequencing platforms hold promise for expanding our understanding of DNA replication dynamics. Integrating strand-specific information into single-cell studies could offer novel insights into enzymology, opening avenues for future research and applications in repair and replication biology.