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bioRxivCell BiologyPreprint

2C-RT Quantifies Genome-Wide DNA Replication Perturbations and Redefines RIF1-CDC7 Regulation of Replication Timing

Bruno UriéniD, Philip Yuk KuwongiD, Giovanni GiglioiD, Carmen NavarroiD, Matilde Rodrigues da FonsecaiD, Sunil PradhaniD, Simon J. ElsässeriD, Bennie B. L. G. LemmensiD

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In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (23 Oct 2026) · CC BY 4.0

Sorting S-phase cells by Cyclin A2 rather than DNA content exposes a uniform genome-wide delay after CDC7 inhibition and a broad replication timing advance in RIF1 knockout.

Replication timing assays infer S-phase position from DNA content, which misleads when DNA synthesis is perturbed and has produced paradoxical RIF1 and CDC7 results. 2C-RT sorts on Cyclin A2, a DNA content-independent timer, to resolve these cases.

Key results

  1. ShownUnder CDC7 inhibition (XL413), Cyclin A2 still tracks S-phase progression while DAPI-based DNA content does not, so DNA-content gating masks the delay in replication onset.

    In RPE cells, XL413 shifted S-phase cells toward high Cyclin A2 but gave no clear DAPI shift. In Palbociclib release time courses (2 to 12 h), Cyclin A2 and Geminin kinetics matched control, while EdU and DNA content lagged. FACS gating on Cyclin A2 showed a late-S shift. · Cyclin A2 reveals replication delays masked by…

  2. Shown2C-RT and classic DNA-content RT agree in unperturbed cells but diverge after CDC7 inhibition: 2C-RT reports a near-universal delay, while classic RT reports a mixed pattern with apparent late-region advance.

    Mock 2C-RT vs classic RTI Pearson r = 0.97 (50-kb bins). With XL413, 93.22% of bins were delayed in 2C-RT vs 37.79% in classic RT. Late-replicating bins (RTI > 0.75) matched H3K9me3, lamina proximity and peripheral GPSeq positions. · Development and validation of 2C-RT

  3. ShownRIF1 loss advances replication across most of the genome rather than randomizing timing, and heterochromatic late regions also advance, contrary to the classic RT reading that they escape RIF1.

    RIF1KO vs p53KO RTI correlation r = 0.54 in both methods. 2C-RT RIF1KO data sit 0.16 RTI units earlier than classic RT. Classic RT implied nearly half the genome was delayed, while 2C-RT showed only a minor early-S delay. One RIF1KO line. · 2C-RT detects genome-wide and local changes in…; 2C-RT reveals a general RT advance in RIF1KO…

  4. SuggestedRIF1-dependent advances are larger toward the nuclear periphery and in gene-sparse chromosomes, and pulse-chase imaging supports altered peripheral replication patterns in RIF1KO cells.

    In 2C-RT, chromosome-level ΔRTI correlated with GPSeq position, and the largest advances were in low gene density chromosomes (correlation values are in figures only). Triple-positive cells imaged: N = 285, 852, 325, 586 (p53KO mock, p53KO CDC7i, RIF1KO mock, RIF1KO CDC7i). · 2C-RT reveals a general RT advance in RIF1KO…; Figure legends

  5. SuggestedThe MCM5 bob1 (P92L) mutation reduces early-site dominance under CDC7 inhibition, with the central peak flanked by two equal side peaks, and it appears to lower the CDK2 activity threshold, since mutant cells resist CDK2 inhibition.

    Across 304 early initiation sites, XL413-treated bob1 cells showed a central peak flanked by two equal peaks about 750 kb away, while control cells kept a dominant central peak. After a 30-minute NU6140 pulse, more cells stayed EdU-positive in bob1 than in MCM5 wild-type cells, mostly in mid/late S-phase. · 2C-RT uncovers genetic and temporal control of…; 2C-RT detects genome-wide and local changes in…

Methods, data and reproducibility

Cells are sorted by EdU plus Cyclin A2 (or DAPI) into early, mid and late S gates, barcoded and pooled by MINUTE-ChIP, and EdU-DNA is sequenced. A Replication Timing Index (RTI) per 50-kb bin is compared across drugs and genotypes, with QIBC and 3D-SIM imaging.

  • RPE p53KO parental, RIF1KO and MCM5 bob1 lines, mock vs XL413, 2C-RT and classic RT sequencing · human (hTERT-RPE-1) · n = one replicate per sort (Fig 2B legend)
  • Early initiation zones defined from mock MCM5 wild-type 2C-RT data · human · n = N = 304 sites
  • 3D-SPARK super-resolution nascent DNA imaging, MCM5 wild-type vs bob1 clones · human · n = N = 16 nuclei per condition (14 for clone #11B)
  • QIBC in RPE, U2OS and BJ cells after XL413, NU6140 or Palbociclib release · human · n = more than 1,000 cells per replicate and condition
  • Published RPE datasets: H3K9me3, RTI, chromatin states, Lamin B1 TSA-seq, GPSeq · human
Methods & tools
2C-RT, MINUTE-ChIP, QIBC, 3D-SPARK (3D-SIM), CellProfiler, snakemake pipeline (minute), wigglescout, UCSC LiftOver, Jalview MUSCLE, Benchling, FACSAria Fusion, Illumina NextSeq 2000
Software & environment
Reads mapped to hg38; Jalview 2.11.5.2 (MUSCLE); ZEN 2011 SP2 Black for 3D-SIM reconstruction; custom snakemake pipeline and wigglescout for binning.
Reproducibility gaps
No sequencing accession or data repository is stated, and no dedicated code for RTI or 2C-RT downstream analysis is given. The MCM5 bob1 clones were made for this study.
Limitations (3)
  • Geminin-based timing (Gem1-110-mCherry) saturates after early S-phase, so it is confined to early S-phase quantification, whereas Cyclin A2 is the broader timer. · stated by the authors
  • Sequencing comparisons rest on one replicate per sort and single RIF1KO and p53KO lines, so no replicate-level variance for RTI or ΔRTI is reported; the proposed redistribution of limiting firing factors in RIF1KO is untested. · noted in this summary
  • The proposed lowering of the CDK2 activity threshold by bob1 rests on a 30-minute NU6140 inhibitor test read out by QIBC, with no direct biochemical measurement of CDK activity. · noted in this summary

Read this if you profile replication timing under drugs or mutants and need to know how DNA-content gating can bias RT conclusions about RIF1, CDC7 or MCM.

Citations
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Abstract

Replication timing (RT) coordinates DNA synthesis with genome organization and influences mutation rates, cancer evolution, and developmental plasticity. Current methods infer S-phase timing from DNA content, an assumption that obscures responses to replication perturbations and limits accurate comparisons of RT changes. Here, we develop 2C-RT, a multiplexed sequencing approach that uses DNA content-independent markers for robust RT profiling across S phase. Combining 2C-RT with imaging, we resolve distinct layers of human RT control and identify local and genome-wide responses to drugs and genetic perturbations. We find that RIF1 spatially restricts CDC7 activity and that its loss does not neutralize RT per se but advances replication across most RIF1-sensitive regions. A conserved MCM5 bob1 mutation lowers the origin-activation threshold and reveals a role for CDC7 in establishing local origin dominance. These findings support a revised model in which CDC7 primes early origins, CDK promotes permissive origin firing genome-wide, and RIF1 counteracts CDC7 in distinct chromatin states and peripheral nuclear domains.

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