Characterizing and controlling intrinsic biases of Lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins

Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of Lambda exonuclease (?-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We took genomics and biochemical approaches to determine if ?-exo digests all parental DNA sequences equally. We report that ?-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, ?-exo digestion of non-replicating genomic DNA (LexoG0) enriches GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand independent ?-exo biases in NS-seq and validated this approach at the rDNA locus. The ?-exo-controlled NS-seq peaks are not GC-rich and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification, but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na+ instead of K in the ?-exo digestion buffer reduced the effect of Na G4s on ?-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq