# Mapping the 3D architecture of native human replisomes

> **NIH NIH R01** · SAN DIEGO BIOMEDICAL RESEARCH INSTITUTE · 2021 · $592,081

## Abstract

ABSTRACT
DNA replication is central to human genome integrity and is intimately tied to large-scale 3D genome architecture
and cell lineage specification, yet we still do not have reliable maps of replicon organization nor any molecular
tools to study how dismantling and re-assembly of 3D architecture is executed and coordinated with transcription.
Our long-term goal is a complete understanding of the 3D choreography of replication over the course of S phase
and its coordination with transcription. The overall objective of this application is to obtain direct measurements
of replicon organization during S phase and model their 3D organization. Our central hypothesis is that replication
initiation occurs stochastically at several (of many) potential origins that are in close 3D proximity at the time of
initiation, after which forks remain in close proximity as chromatin transiently disengages from transcription and
interphase 3D interactions. Our rationale is that high resolution single molecule 3D maps of nascent DNA will
uncover novel mechanistic insights into how replication is faithfully executed and coordinated with transcription.
AIM1 will develop a transformative single DNA fiber optical replication mapping (ORM) method, permitting us to
map origins and fork polarities on single molecules with unprecedented throughput (30Gb/hr). We will integrate
these maps with high resolution Repli-seq and Hi-C maps to reveal how replicons are organized in time and
space. To model the native 3D structure of individual replisomes, we will develop replication fork-enriched
versions of single-particle SPRITE (split pool recognition of interactions by tag extension), Hi-C and single cell
Hi-C. SPRITE enables detection of multiple simultaneously occurring DNA and RNA interactions within cross-
linked and individually bar-coded large chromatin complexes. In AIM2, we will capture complexes containing
pulse-labeled nascent DNA (Repli-SPRITE) to assess 3D association of DNA and RNA, including nascent RNA,
with active replication forks (i.e. replisomes). In AIM3, we will map DNA in close proximity to active replication
forks by capturing pulse-labeled nascent DNA from Hi-C libraries (Repli-Hi-C). Population Repli-Hi-C will provide
a high resolution global view of how contacts differ as replication forks pass through domains, while single cell
Repli-Hi-C will enable 3D models of how multiple replicons are organized within domains and how replication is
temporally coordinated across the genome in each cell. Importantly, AIMs 2 and 3 will also chase the labeled
DNA before capture to track the dynamic re-assembly of interphase 3D structures. We expect to deliver an
unprecedented view of how the human genome is organized for DNA replication and how replication is
coordinated with 3D architecture and transcription. This contribution will be significant because it will deepen our
understanding of how DNA replication is orchestrated to preserve genome integrity and cell-ty...

## Key facts

- **NIH application ID:** 10400294
- **Project number:** 7R01HG010658-03
- **Recipient organization:** SAN DIEGO BIOMEDICAL RESEARCH INSTITUTE
- **Principal Investigator:** David M Gilbert
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $592,081
- **Award type:** 7
- **Project period:** 2019-08-02 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10400294

## Citation

> US National Institutes of Health, RePORTER application 10400294, Mapping the 3D architecture of native human replisomes (7R01HG010658-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10400294. Licensed CC0.

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