# DNA Replication Initiation Sites in Mammalian Cells

> **NIH NIH R01** · ALBERT EINSTEIN COLLEGE OF MEDICINE · 2020 · $604,542

## Abstract

PROJECT SUMMARY / ABSTRACT
Repetitive DNA sequences often challenge DNA replication, which can lead to genomic instability. Aberrant
replication of two repeat-rich regions in particular, telomeres and common fragile sites (CFS), have
pathological consequences. The goal of this proposal is to elucidate mechanisms that ensure normal telomere
and CFS replication and protect genomic integrity. Replication fork stalling at repetitive sequences is thought to
be a main causative factor underlying both telomere- and CFS-associated genomic instability. Using our single
molecule approach, termed SMARD (single molecule analysis of replicated DNA), we have shown that stalling
occurs at both telomeres and CFS. Therefore we propose to examine how stalling is overcome at these
important chromosomal elements with the following aims. In Aim1, we will establish the role of replication
initiation within telomeres in resuming stalled replication and maintaining proper telomere function. Initiation of
replication ahead of a stalled fork is a key replication recovery response. We will disrupt replication fork
progression in human and mouse cells using replication inhibitors, G-quadruplex stabilizers and replication
barriers, and examine telomere replication to establish that telomeric initiation will occur to rescue stalled
replication. Key events driving telomere-associated genetic instability are telomere shortening and failure to
suppress DNA damage responses and deleterious repair (telomere dysfunction). We will determine if reduced
or compromised telomeric initiation leads to defective telomere replication resulting in telomere loss and
dysfunction, to establish whether telomeric initiation provides a protective mechanism for maintaining proper
telomere function. In Aim 2, we will establish mechanism(s) that facilitate the replication of stall-prone regions
in CFS loci. Our preliminary studies reveal that the Fanconi anemia complementation group D2 protein
(FANCD2) plays an important role in facilitating CFS replication by alleviating replication pausing. Thus, we will
perform studies to further define its role in CFS replication. Since FANCD2 interacts with translesion
polymerases, which have been implicated in CFS replication, we will establish whether FANCD2 recruits these
polymerases to aid in CFS replication. We will also investigate whether FANCD2 alleviates transcription-
associated obstacles to replication fork progression including RNA: DNA hybrids, because FANCD2–deficient
cells display an increased abundance these hybrids genome-wide. Our preliminary studies reveal that changes
in replication origin usage are associated with FANCD2 deficiency. Because altered origin usage is linked to
changes in chromosome organization, we will determine if FANCD2 promotes a chromosome architecture that
facilitates accurate CFS replication. We expect that these proposed studies will both greatly increase our
understanding of telomere and CFS replication and allow us to...

## Key facts

- **NIH application ID:** 9870931
- **Project number:** 5R01GM045751-29
- **Recipient organization:** ALBERT EINSTEIN COLLEGE OF MEDICINE
- **Principal Investigator:** CARL L SCHILDKRAUT
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $604,542
- **Award type:** 5
- **Project period:** 1992-01-08 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9870931

## Citation

> US National Institutes of Health, RePORTER application 9870931, DNA Replication Initiation Sites in Mammalian Cells (5R01GM045751-29). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9870931. Licensed CC0.

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