Southeastern Regional Yeast Meeting (SERYM) 2022

Abstract

Ribonucleotide (rNMP) incorporation in DNA generally occurs in all kingdoms of life, results in DNA structural change, genome instability, and influences the protein-DNA interaction. Previous studies in yeast showed that replicative DNA polymerases α, δ, and ε, particularly low-fidelity mutants of these Pols misincorporate substantial rNMPs in DNA during DNA replication. These rNMPs are efficiently removed with the help of ribonuclease (RNase) H2. In this study, we performed a computational analysis of rNMPs embedded around yeast autonomously replicating sequences (ARSs), where DNA replication starts, and Pols α, δ, and ε synthesize the leading and lagging strands. We used published rNMP incorporation datasets generated by ribose-seq, emRiboSeq, and RHII-HydEn-seq techniques. We analyzed the rNMP incorporation strand biases in both wild-type and ribonuclease (RNase) H2-nulllibraries. The results show an overall preference of rNMP incorporation on the leading strand in wild-type Pols 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 ARSs 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 uncover the characteristics of rNMP incorporation around ARS’s, which are induced by different DNA polymerases, and we validate the labor division of DNA polymerases. This work is supported by NIH, NIEHS R01 ES026243, and Howard Hughes Medical Institute Faculty Scholars Award, HHMI 55108574 to F. Storici.