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 (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-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 λ-exo biases in NSseq and validated this approach at the rDNA locus. The λ-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 λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq.

The chromatin environment shapes DNA replication origin organization and defines origin classes

“…As a control, we also used genomic DNA sonicated to the same size as the NS DNA and treated by λ−Exo as described for the NS samples. This protocol differs in several ways from a recently described method (Foulk et al 2015) in which short NS were not purified by sucrose gradient before λ−Exo digestion. Instead, a large amount of whole replicating DNA was used.…”

DNA replication origins—where do we begin?

“…Size-selected nascent strands were enriched and purified from broken contaminating chromosomal fragments by treatment with excess λ-exonuclease before hybridization to microarrays. It should be noted that excess λ-exonuclease is important for the accurate identification of bona fide nascent strands, as the nuclease exhibits an intrinsic sequence bias that can lead to false positives (Foulk et al 2015). Despite surveying only a limited portion of the genome, it was possible to estimate a population-based average interorigin distance of ∼60 kb (Cadoret et al 2008).…”

Replication origins: determinants or consequences of nuclear organization?

“…Although mammalian replication origins do not share a single “consensus” sequence, a large fraction of origins is located adjacent to transcriptional start sites (TSSs), regions of DNase hypersensitivity [11,24,27–30] and G-rich sequences, including CpG islands and sequences that can potentially form G-quadruplex structures [11,24,27,30]. It is unclear whether these colocalizations represent causal relationships.…”