# Mechanistic studies of stalled DNA replication fork rescue

> **NIH NIH R01** · UNIVERSITY OF NEBRASKA MEDICAL CENTER · 2021 · $60,174

## Abstract

ABSTRACT OF THE PARENT GRANT GM10056
There is a fundamental gap in understanding how stalled DNA replication forks are rescued. The continued existence of
this gap represents an important problem because, until it is filled, a complete and clear understanding of the mechanism of
stalled fork reactivation will be lacking. This understanding is crucial as defects in these repair mechanisms in higher
organisms lead to the accumulation of mutations leading to cancer, and the proposed studies are therefore directly relevant
to human disease. Consequently, the long-term goal is to understand the mechanism of stalled DNA replication fork
reactivation. The main objective of this proposal is to understand the interplay between the single-stranded DNA binding
protein (SSB) and key fork rescue enzymes on nucleoid templates and of the subsequent processing events leading to
restoration of a fork structure. To achieve this objective, this proposal is divided into three specific aims: 1), Determine the
mechanism(s) of fork regression; 2,) To determine how fork impediments affect fork regression; and 3), Ascertain the effects
of nucleoid-associated proteins on fork rescue enzymes. Under the first aim, magnetic tweezers and atomic force microscopy
(both in air and high-speed in buffer) will be used to determine how SSB loading and regression by RecG are affected by
PriA and to ascertain whether RecA and RuvAB are able to catalyze an efficient and unidirectional fork regression reaction.
When the proposed studies for Aim 1 are complete, a clear picture of the events at a nascent, stalled replication fork will be
provided. Under the second aim, the same two single DNA molecule approaches will be used to provide insight into the
effects of replisome impediments on stalled fork rescue, with high spatial and temporal resolution. At the conclusion of the
proposed studies for Aim 2, the effects of DNA lesions and protein-DNA complexes on fork rescue will be made clear and
it is anticipated that the mechanism(s) for displacing stalled RNA polymerase in the vicinity of forks will be obtained. Under
the final aim, magnetic tweezers to manipulate single molecules of DNA will be used to ascertain the effects of nucleoid-
associated proteins (NAPs) on fork rescue. When the proposed studies for Aim 3 are complete, it will be ascertained whether
NAPs catalyze regression on their own and if they assist or inhibit fork rescue enzymes. The proposed research is innovative
because of the combinatorial strategy taken. It is also innovative because of the exciting and novel single-molecule
approaches used, the focus on nucleoid templates, and an understanding to be gained of how the primary protein barrier(s)
causing replisome stalling are removed. Finally, the work is also innovative because of the care taken in elucidating how
recombination helicases function in the presence of SSB. The proposed research is significant because it will allow, for the
first time, the development of c...

## Key facts

- **NIH application ID:** 10387612
- **Project number:** 3R01GM100156-09S1
- **Recipient organization:** UNIVERSITY OF NEBRASKA MEDICAL CENTER
- **Principal Investigator:** Piero R Bianco
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $60,174
- **Award type:** 3
- **Project period:** 2013-06-07 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10387612

## Citation

> US National Institutes of Health, RePORTER application 10387612, Mechanistic studies of stalled DNA replication fork rescue (3R01GM100156-09S1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10387612. Licensed CC0.

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