# Control of topoisomerase activity during DNA replication by bacterial chromosome structuring proteins

> **NIH NIH K99** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $99,999

## Abstract

DNA replication is stressful because unwinding of the DNA strands by the replication fork generates highly
structured, supercoiled DNA that must be removed or else the replication fork will stall. DNA topoisomerases
are ubiquitous enzymes that are essential for relaxing the supercoiling barriers formed during DNA replication.
Although the mechanisms of how topoisomerases relieve DNA strain have been well-described, how these
enzymes are regulated in vivo and how they are localized to specific regions of the genome, e.g., ahead of
replication forks, remains poorly understood. As therapeutics that target topoisomerases are used to treat
disorders such as cancer and bacterial infections, a better understanding of these enzymes will be key for
future drug development. My postdoctoral studies identified an essential chromosome structuring protein
called GapR in the bacterium Caulobacter crescentus that is required for replication and specifically
recognizes overtwisted DNA, such as the DNA ahead of replication forks. Critically, GapR also stimulates the
activity of bacterial topoisomerases, gyrase and topo IV, to relax highly supercoiled DNA by an unknown
mechanism. This proposal will examine new regulatory paradigms by investigating how chromosome
structuring proteins such as GapR control topoisomerase activity during replication. Aim 1 will elucidate the
mechanisms by which GapR stimulates topoisomerase activity. I will perform co-immunoprecipitation assays to
determine if GapR interacts with topoisomerases to stimulate their activities. Alternatively, or in addition, GapR
could trap supercoiling by binding overtwisted DNA and consequently allow these trapped supercoils to be
more efficiently relaxed by gyrase and topo IV. I will test this idea by examining how GapR interacts with DNA
using magnetic tweezers. Lastly, I will use magnetic tweezers to directly assess how GapR stimulates the
topoisomerase catalytic cycle. Aim 2 will examine how GapR regulates topoisomerase localization and activity
to promote replication. I will determine how loss of GapR affects topoisomerase binding with ChIP-seq and
develop assays to examine topoisomerase activity genome-wide by sequencing the DNA trapped in
catalytically active topoisomerases. I will also assess how loss of GapR affects replication fork progression,
particularly in regions that have hyper supercoiling, by examining binding of replicative helicase with ChIP-seq.
In Aim 3, I will search for additional, GapR-like topoisomerase co-factors using mass spectrometry and
transposon-based screens. I will subsequently characterize any identified co-factor proteins with biochemical,
biophysical, and genetic approaches. The experiments within these aims will be initiated during the K99 phase
of the award and will include training with magnetic tweezers to study topoisomerase mechanism and high-
throughput approaches to study topoisomerase activity in vivo. Together, the experiments in this proposal will
describe...

## Key facts

- **NIH application ID:** 9978843
- **Project number:** 5K99GM134153-02
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Monica S. Guo
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $99,999
- **Award type:** 5
- **Project period:** 2019-07-16 → 2021-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9978843

## Citation

> US National Institutes of Health, RePORTER application 9978843, Control of topoisomerase activity during DNA replication by bacterial chromosome structuring proteins (5K99GM134153-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9978843. Licensed CC0.

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