# Mechanism of Microbial DNA Hypervariation through Mutagenic Transposition

> **NIH NIH R01** · UNIVERSITY OF MISSOURI-COLUMBIA · 2020 · $288,714

## Abstract

Project Abstract
Diversity-generating retroelements (DGRs) are molecular evolution machines found in bacteria,
archaea and their viruses. They diversify protein-encoding sequences to facilitate the adaptation of
their hosts to changing environments. Hypervariation results from an error-prone retrotransposition
process called mutagenic homing, which transfers sequence information from a template repeat (TR)
to a variable repeat (VR) that results in adenine to random nucleotide conversions. The long-term goal
of the PI’s group is to understand the mechanism of DGR mutagenic homing and to develop them for
practical applications. In analogy to related retroelements, DGR homing was proposed to occur
through a target DNA-primed reverse transcription mechanism. Interestingly, recent discoveries by the
PI’s group showed that reverse transcription of TR of the Bordetella phage DGR is primed by a
downstream adenine residue of the RNA intermediate and is target (VR)-independent. Intriguingly, the
TR RNA intermediate was found to be nicked in a bRT (Bordetella reverse transcriptase)-dependent
manner to generate a 3’-OH for cDNA priming, and single amino acid substitutions at the RT catalytic
core abolish the nicking activity, suggesting that bRT plays a catalytic role in the cleavage reaction.
Adenine-specific mutagenesis occurs during (–)cDNA synthesis and results from misincorporation of
standard deoxyribonulceotides by bRT. In addition, mutational analysis showed that this special,
target-independent reverse transcription reaction is responsible for DGR mutagenic homing, revealing
a novel mechanism of DNA hypervariation. The specific aims are based on these novel discoveries.
Aim 1 will characterize the secondary structure of TR RNA and map the Avd and bRT binding sites on
the RNA intermediate. These studies will generate the first secondary structure of a DGR RNA, and
reveal whether the TR RNA intermediate is catalytically cleaved by bRT. Aim 2 will determine the
mechanism of cDNA integration at the 3’ and 5’ ends of VR. Roles of base pairing interactions
between the RNA primer and VR DNA and between cDNA and VR DNA in 3’ cDNA integration will be
tested. These studies may lead to discovery of novel cDNA integration mechanisms. Aim 3 will
determine the mechanism of adenine-specific mutagenesis, which is a hallmark of DGRs.
Understanding the mechanism of adenine-specific mutagenesis will likely yield new insights on RT
fidelity issues. In summary, studies proposed in this application will elucidate the mechanism of DGR
mutagenic homing, which may have broad implications in health and science.

## Key facts

- **NIH application ID:** 9984865
- **Project number:** 5R01GM125669-03
- **Recipient organization:** UNIVERSITY OF MISSOURI-COLUMBIA
- **Principal Investigator:** Donald H Burke
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $288,714
- **Award type:** 5
- **Project period:** 2018-09-19 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9984865

## Citation

> US National Institutes of Health, RePORTER application 9984865, Mechanism of Microbial DNA Hypervariation through Mutagenic Transposition (5R01GM125669-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9984865. Licensed CC0.

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