# Structure and function of DNA polymerase lambda opposite DNA lesions which disrupt Watson-Crick base pairing

> **NIH NIH R01** · UNIVERSITY OF TEXAS MED BR GALVESTON · 2021 · $254,460

## Abstract

DNA polymerase (Pol) , a B-family Pol, plays an important role in replication of damaged DNA. Extensive
biochemical studies with Pol have shown that it is very inefficient at inserting nucleotides (nts) opposite DNA
lesions but is highly efficient at extending synthesis from the correct or incorrect nt opposite DNA lesions.
Since translesion DNA synthesis (TLS) through a large variety of DNA lesions requires the sequential action of
an inserter and an extender Pol, by extending synthesis opposite from diverse DNA lesions, Pol performs a
critical task in the replication of damaged DNA. In yeast, Rev1 performs an indispensable but non-catalytic role
as a component of Pol and it increases Pol’s efficiency for extension from incorrect nts opposite DNA
lesions. Consequently, the Rev1/Pol complex promotes highly error-prone TLS and thereby accounts for
damage induced mutagenesis in yeast. In striking contrast to the requirement of Rev1 for yeast Pol function,
Rev1 is not required for Pol function in TLS in normal human cells. Instead, we provide evidence here that
Pol acts as an indispensable component of human (h) Pol and in concert with Pol, hPol promotes a
predominantly error-free mode of TLS opposite various DNA lesions. In the proposed studies, we will utilize a
combination of genetic, cellular, biochemical, and structural studies to: elucidate the role of Pol in Pol
dependent TLS in human cells, determine the fidelity and action mechanism of Pol in TLS opposite DNA
lesions which impair Watson-Crick (W-C) base pairing, and define the molecular mechanisms that allow Pol’s
active site to handle such DNA lesions. In Aim 1, we will: (a) analyze the requirement of Pol and its domains
for Pol-dependent TLS and mutagenesis opposite a variety of DNA lesions in human cells; (b) examine the
role of Pol in UV induced mutagenesis in the cII gene carried in the mouse genome; (c) determine the
requirement of Pol domains for localization of Polinto replication foci in UV damaged human cells; (d)
examine whether Pol associates with hPol in a physical complex in UV damaged human cells; and (e)
determine the effects of Pol on fork progression in UV irradiated human cells. In Aim 2, we will (a) use
steady-state kinetic analyses to determine the catalytic efficiency and fidelity of Pol in inserting nucleotides
opposite N1-methyladenine (N1-MeA) and the 3’T and 5’T of a (6-4) TT photoproduct, and (b) carry out pre-
steady-state kinetic studies to determine the action mechanism of Pol for inserting the correct nt opposite
these DNA lesions which impair W-C base pairing. In Aim 3, we will (a) determine the structures of binary and
ternary complexes of Pol bound to N1-MeA template in the absence or presence of an incoming dTTP,
respectively, (b) determine the structures of binary and ternary complexes of Pol bound to the (6-4) TT
photoproduct-containing templates in the absence or presence of an incoming dATP, respectively, and (c)
carry o...

## Key facts

- **NIH application ID:** 10065004
- **Project number:** 5R01GM126087-04
- **Recipient organization:** UNIVERSITY OF TEXAS MED BR GALVESTON
- **Principal Investigator:** LOUISE PRAKASH
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $254,460
- **Award type:** 5
- **Project period:** 2017-12-18 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10065004

## Citation

> US National Institutes of Health, RePORTER application 10065004, Structure and function of DNA polymerase lambda opposite DNA lesions which disrupt Watson-Crick base pairing (5R01GM126087-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10065004. Licensed CC0.

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