# Mechanisms of Copying of Carcinogen-damaged DNA and RNA by Translesion Polymerases

> **NIH NIH R01** · VANDERBILT UNIVERSITY · 2021 · $354,778

## Abstract

PROJECT SUMMARY
 Genetic integrity is important in the maintenance of cellular homeostasis. Chemical and physical
damage is well-known with DNA but less extensively studied with RNA. Miscopying of damaged bases is
known to contribute to mutations and to cancer and other diseases. A focus in this laboratory for more than
two decades has been DNA polymerases and their interactions with damaged DNA. Recent studies in our
lab and by others have shown that DNA polymerases sometimes incorporate ribonucleoside triphosphates
(rNTPs). In this laboratory, at least two of the human (h) translesion DNA polymerases, hpol η and hpol κ,
have been shown to have unexpected activities with both DNA and RNA templates, including reverse
transcription, DNA priming, and transcription. These activities will be studied systematically, particularly
with several common lesions known to be formed in DNA and hypothesized to also be present in RNA. It is
hypothesized that specific steric and bonding features of these polymerases impart these novel properties
and that these biological properties are operative in cells. Features of the proposed studies include: (i)
Characterization of the activity of hpol η in transcription, reverse transcription, and DNA priming. Steady-
state and pre-steady-state kinetic analysis will be done to identify catalytic specificity and rate-limiting
reaction steps. A key feature of the work will be several adducts in DNA and RNA (7,8-dihydro-8-oxoG (8-
oxoG), 1,N6-ethenoadenine, and (thymidine-thymidine) cyclobutane pyrimidine dimer (CPD)), including
analysis of how hpol η inserts correct or incorrect bases opposite each of these in RNA templates. X-Ray
crystallography will be used to define features of hpol η contributing to the observed catalytic properties.
Another feature of the work is analysis of levels of individual RNA adducts. (ii) Some similar questions will
be addressed with hpol κ, which has been demonstrated to have reverse transcription and DNA priming
activities. hpol ι has been reported to be capable of inserting rNTPs opposite undamaged DNA, an abasic
site, and 8-oxoG. The mechanism will be investigated using X-ray crystal structures of ternary complexes
with native and adducted DNA templates. (iii) The biological relevance of the hpol η reactions with RNA
templates and insertion of ribonucleotides into DNA will be tested by investigating ribonucleotide insertion by
hpol η opposite CPD (considered the most natural substrate for hpol η) under physiological conditions
including typical cellular concentrations of metals and ribo- and 2ʹ′-deoxyribo-nucleotides. The effect of
ribonucleotides opposite CPD will be examined with RNase H2 and nucleotide excision repair systems, in
that CPD levels have been related to the presence of ribonucleotides in DNA, which in term are linked to
systemic autoimmunity. XPV fibroblast cell extracts and cells will be utilized, which differ from control cells in
whether they contain hpol η. Collectively t...

## Key facts

- **NIH application ID:** 10124392
- **Project number:** 5R01ES026955-05
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** F PETER Guengerich
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $354,778
- **Award type:** 5
- **Project period:** 2017-04-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10124392

## Citation

> US National Institutes of Health, RePORTER application 10124392, Mechanisms of Copying of Carcinogen-damaged DNA and RNA by Translesion Polymerases (5R01ES026955-05). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10124392. Licensed CC0.

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