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.

ABSTRACT: 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 used 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 nonreplicating 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 G4s on ?-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq.

SUBMITTER: Foulk MS 

PROVIDER: S-EPMC4417120 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature