2021 Molecular Bio-Medical Research Community Seminar Series
Abstract
Ribonucleotide (rNMP) incorporation in DNA generally occurs in all kingdoms of life, results in DNA structural change and genome instability. Previous studies in yeast showed that the rNMP presence in DNA is strongly induced by misincorporation of replicative DNA polymerases α, δ, and ε, particularly with low-fidelity mutants of these Pols. rNMP presence in DNA is more evident in cells with defects in ribonuclease (RNase) H2, which normally initiates rNMP removal from genomic DNA. Using published rNMP incorporation datasets generated by ribose-seq, emRiboSeq, and RHII-HydEn-seq techniques, we performed a computational analysis of rNMP sites around yeast autonomously replicating sequences (ARSs), where DNA replication starts, and Pols α, δ, and ε synthesize the leading and lagging strands. We analyzed the rNMP incorporation strand biases in both wild-type and RNase H2-mutant libraries. The results show an overall preference of rNMP incorporation on the leading strand in wild-type Pol and Pol ε low-fidelity mutant libraries. But Pol α or Pol δ low-fidelity mutant libraries display a preference for rNMP incorporation on the lagging strand. All the rNMP preferences are reduced around late-firing and low-efficiency ARS’s in RNase H2-mutant libraries. Furthermore, at the beginning of DNA replication, the leading/lagging-strand ratio of rNMP incorporation increases in wild-type DNA Pol and Pol ε low-fidelity mutant libraries and decreases in Pol δ low-fidelity mutant libraries, which reflects the Pol δ- Pol ε handoff and validates replicative polymerase division of labor in the leading strand synthesis. Moreover, we found the different rNMP incorporation context preferences of different DNA polymerases. The DNA Pol δ prefers to incorporate rNMPs after dCMP while Pol ε prefers to incorporate rNMPs after dCMP. Those preferences are strengthened with low-fidelity mutants. Overall, we found the characteristics of rNMP incorporation around ARS’s, which are induced by different DNA polymerases, and validate the labor division of DNA polymerases.
