title: 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 NS-seq 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.
authors.affiliation: Brown University Division of Biology and Medicine, Department of Molecular Biology, Cell Biology and Biochemistry, Providence, Rhode Island 02912, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Foulk
authors.affiliation: Brown University Division of Biology and Medicine, Department of Molecular Biology, Cell Biology and Biochemistry, Providence, Rhode Island 02912, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Urban
authors.affiliation: Brown University Division of Biology and Medicine, Department of Molecular Biology, Cell Biology and Biochemistry, Providence, Rhode Island 02912, USA.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Casella
authors.affiliation: Brown University Division of Biology and Medicine, Department of Molecular Biology, Cell Biology and Biochemistry, Providence, Rhode Island 02912, USA.
authors.forename: Susan A
authors.initials: SA
authors.lastname: Gerbi
mesh_terms: D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D054856:G-Quadruplexes; D020862:GC Rich Sequence; D006801:Humans; D061986:MCF-7 Cells; D009707:Nucleosomes / Q000737:chemistry* / Q000235:genetics; D018741:Replication Origin; D017422:Sequence Analysis, DNA / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004275:DNA, Ribosomal; D009707:Nucleosomes; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda