# The role of DNA damage tolerance pathways in human cells

> **NIH NIH R01** · UNIVERSITY OF MINNESOTA · 2022 · $294,297

## Abstract

Project Summary
Accurate DNA replication is essential for preserving genomic stability and protecting
against carcinogenesis. Obstacles to DNA replication, such as DNA adducts, secondary
DNA structures, or nucleotide depletion block the progression of DNA polymerases and
thus arrest replication forks, which can lead to fork collapse and DNA breaks. As this is a
source for point mutations and structural variations, understanding replication stress is
important for both cancer prevention and treatment. Stalled replication forks can be
reversed, a process which stabilizes stalled forks, but also exposes them to nucleolytical
degradation, unless protected through loading of RAD51 by the FA-BRCA pathway.
 PCNA is an essential replication fork component, which upon exposure to DNA
damage is ubiquitinated at lysine (K) 164. This event controls two mechanisms of DNA
lesion bypass, translesion synthesis and template switching. While traditionally PCNA
mono-ubiquitination was considered a DNA damage-induced event, recent work by our
laboratories and others suggested a more broad impact of PCNA ubiquitination during
replication. To mechanistically address this, we employed the CRISPR/Cas9 genome
editing technology to introduce the K164R mutation in the endogenous PCNA alleles in
HEK293T, RPE1, and HCT116 cells. Preliminary characterization of these cells revealed
unexpected genomic instability features, including telomere erosion, replication fork
degradation, and gross chromosomal rearrangements. Based on these preliminary
results, we propose that PCNA ubiquitination has previously unrecognized roles in
regulating genomic stability. We will address this in three specific aims:
 Aim 1 will investigate the role of PCNA modification at K164 in telomere
maintenance. Aim 2 will investigate the role of K164-modified PCNA in suppressing
replication fork degradation. Aim 3 will elucidate the functional impact of PCNA
modification at K164 on genomic rearrangements. Our work is poised to uncover novel
cellular mechanisms regulated by PCNA modification at K164, and will have a significant
impact on our understanding of genome integrity.

## Key facts

- **NIH application ID:** 10436922
- **Project number:** 5R01GM134681-04
- **Recipient organization:** UNIVERSITY OF MINNESOTA
- **Principal Investigator:** Anja-Katrin Bielinsky
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $294,297
- **Award type:** 5
- **Project period:** 2019-08-14 → 2022-11-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10436922

## Citation

> US National Institutes of Health, RePORTER application 10436922, The role of DNA damage tolerance pathways in human cells (5R01GM134681-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10436922. Licensed CC0.

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