# Origin firing at repetitive sequences and genome replication - Admin Supplement

> **NIH NIH R01** · FRED HUTCHINSON CANCER CENTER · 2022 · $7,960

## Abstract

Abstract of the Parent Grant (R01 GM117446)
 Over half of the human genome is comprised of repetitive DNA sequences
organized as gene-poor, late replicating, transcriptionally silent heterochromatin.
Recent studies have discerned widespread transcriptional de-repression at repeated
regions during carcinogenesis and aging. De-repression accelerates replication of these
regions and thus compromises replication in the gene-rich transcriptionally active
chromatin. Despite the importance and prevalence of the association between low levels
of transcription and late replication, the mechanistic basis for this link remains unclear.
 The ribosomal DNA (rDNA) as well as copper-inducible CUP1 arrays in
budding yeast are ideal experimental systems in which to elucidate these mechanisms
for two main reasons. First, at each locus, a single manipulation, Sir2 depletion at the
rDNA, and cooper administration at the CUP1, activates both transcription and
replication, providing a simple tool to manipulate both processes. Second, each rDNA
and CUP1 repeat features a single, sequence-defined origin of replication, which creates
uniform and predictable positioning of replication initiation factors and nucleosomes in
their vicinity. This feature makes these systems, and yeast origins in general, ideal for
the application of deep sequencing methods for epigenome profiling we have
developed, offering a tremendous advantage over mammalian origins whose lack of
sequence-specificity confounds modern deep sequencing-based approaches.
Our methods resolves at nucleotide level the precise location of nucleosomes and pre-
replicative complexes (pre-RC) at the repetitive as well as in unique regions of the
genome. Using these methods, we discovered that transcriptional activation at both
rDNA and CUP1 origins leads to reduced occupancy of nucleosomes adjacent to pre-
RC, though by different means at the two loci: At CUP1, transcription reduces
nucleosome occupancy next to the pre-RC loading site, whereas at the rDNA the
nucleosome occupancy next to the pre-RC loading site does not change; instead, RNA
Pol-II pushes pre-RC to an adjacent region with low nucleosome density. Using this
experimental system and the tools for chromatin profiling we have developed, we will
determine whether high nucleosome occupancy adjacent to pre-RC inhibits replication
initiation and whether chromatin compaction-mediated inhibition of nucleosome
remodelers enforce late replication in transcriptionally silent chromatin.

## Key facts

- **NIH application ID:** 10626663
- **Project number:** 3R01GM117446-07S1
- **Recipient organization:** FRED HUTCHINSON CANCER CENTER
- **Principal Investigator:** Antonio Bedalov
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $7,960
- **Award type:** 3
- **Project period:** 2016-02-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10626663

## Citation

> US National Institutes of Health, RePORTER application 10626663, Origin firing at repetitive sequences and genome replication - Admin Supplement (3R01GM117446-07S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10626663. Licensed CC0.

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