# Purification and Initial Biochemical Analysis of the P. aeruginosa ImuABC Error-Prone DNA Polymerase

> **NIH NIH R03** · STATE UNIVERSITY OF NEW YORK AT BUFFALO · 2020 · $75,585

## Abstract

ABSTRACT
 Due to the growing emergence of drug-resistant bacteria, improved therapies, as well as novel targets
against which new therapies can be developed, are desperately needed. A variety of mechanisms contribute to
drug-resistance, and adaptation of pathogens to their human hosts (pathoadaptation), including alterations to
gene expression, the acquisition of new genes, and DNA mutations. A promising therapeutic target that has to
date received remarkably little attention is the role played in pathoadaptation and the acquisition of drug-
resistance by low fidelity DNA polymerases (Pols). These Pols generate mutations when replicating undamaged
DNA, or when bypassing damaged bases via a process termed translesion DNA synthesis (TLS).
 E. coli Pol IV (dinB) and Pol V (umuDC) represent the best-studied bacterial TLS Pols. While most well
studied human pathogens possess a Pol IV homolog that acts in TLS, many lack a Pol V. Instead, they encode
a highly conserved yet understudied multi-subunit TLS Pol that goes by a few different names, and will be
referred to in this proposal as the ImuABC complex. Mutations generated by ImuABC contribute to virulence,
persistence, and drug-resistance. Based on sequence, ImuA (also called ImuA’) has homology to an ATPase
(but likely lacks catalytic activity), while ImuB is a homolog of the Pol V UmuC catalytic subunit that lacks the
essential active site residues, meaning it is likely devoid of Pol activity. Consistent with this conclusion, mutations
catalyzed by ImuABC depend on the Pol activity of ImuC (also called DnaE2), which is structurally related to the
DnaE1 catalytic subunit of the bacterial Pol III replicase. Results of yeast-two-hybrid experiments suggest that
ImuB is an adapter protein that interacts with ImuA and ImuC, as well as the b processivity clamp and the DnaE1
subunit of Pol III. These latter interactions may coordinate the actions of ImuABC with those of Pol III.
 Despite the clear demonstration of an important role for ImuABC in catalyzing mutations that underlie
drug resistance, virulence, and pathoadaptation, the ImuABC complex is the subject of remarkably little research.
At the time of this writing, there were only 42 published papers containing the search terms “imuA, imuB, imuC
or dnaE2.” Importantly, none of these works discuss biochemical analysis of the ImuABC complex. A goal of this
proposal is to develop methods for the purification of soluble forms of the ImuA, ImuB, and ImuC proteins for
detailed in vitro mechanistic studies. For this, we will focus on the P. aeruginosa ImuABC proteins, as we already
have overproducers and established purification methods for the P. aeruginosa b clamp and Pol III replicase,
which will be required in future work aimed at determining the contribution of the ImuB-b clamp and ImuB-Pol III
interactions to ImuABC function/regulation. As a second goal, we will develop several in vitro assays necessary
for detailed biochemical dissection of the mechan...

## Key facts

- **NIH application ID:** 9891550
- **Project number:** 1R03AI144341-01A1
- **Recipient organization:** STATE UNIVERSITY OF NEW YORK AT BUFFALO
- **Principal Investigator:** MARK D. SUTTON
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $75,585
- **Award type:** 1
- **Project period:** 2020-02-03 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9891550

## Citation

> US National Institutes of Health, RePORTER application 9891550, Purification and Initial Biochemical Analysis of the P. aeruginosa ImuABC Error-Prone DNA Polymerase (1R03AI144341-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9891550. Licensed CC0.

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