# Mechanism of NEIL3-dependent ICL repair

> **NIH NIH R00** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2021 · $249,000

## Abstract

During each division, the cell must quickly and accurately replicate its genome. This process,
however, is challenged by constant insults to DNA. DNA interstrand cross-links (ICLs) are
particularly toxic genomic lesions that covalently link the two strands of DNA. If left unrepaired, these
lesions can block replication and induce genomic instability, a hallmark of cancer. Although ICLs
are generated by a variety of exogenous and endogenous agents, the structures of specific ICLs
that arise spontaneously in cells are unknown. In proliferating cells, ICL repair occurs predominately
in S phase. In the classic ICL repair pathway, repair requires replication fork convergence at an ICL
and the cross-linked DNA strands are unhooked by nucleolytic incisions that generate a DNA double
stranded break (DSB) intermediate. This DSB is then repaired by homologous recombination.
Importantly, mutations in genes that function in this repair pathway cause the bone marrow failure
and cancer predisposition syndrome Fanconi anemia (FA). Recently, we discovered an alternative
ICL repair pathway that depends on the NEIL3 DNA glycosylase. Like the FA pathway, the NEIL3
pathway is activated by ubiquitylation of the replicative CMG helicase upon fork convergence at an
ICL. However, unlike the FA pathway, the NEIL3 pathway does not involve formation of a DSB
intermediate. Instead, NEIL3 unhooks ICLs by cleaving one of the N-glycosyl bonds of the crosslinked
nucleobases, generating an abasic site that can be bypassed by translesion synthesis.
Unhooking by the NEIL3 pathway is therefore faster and less complicated than unhooking by the
FA pathway and is the preferred ICL repair pathway for a subset of lesions. In this proposal,
complementary biochemical and analytical approaches will be used to investigate the mechanism
of NEIL3-dependent ICL repair. Aim 1 seeks to determine how replication forks activate NEIL3-
dependent unhooking using Xenopus egg extracts that recapitulate ICL repair. Aim 2 proposes to
investigate the dynamics of NEIL3 at individual replication forks using single molecule approaches.
Finally, Aim 3 will address the question of which endogenous forms of DNA damage are targeted
by ICL repair pathways through the development of a novel mass spectrometry approach to discover
DNA lesions in cells. By understanding the mechanisms of ICL repair, it may be possible to design
interventions that sensitize cancer cells to chemotherapy or mitigate the molecular defects that
cause FA and other diseases.

## Key facts

- **NIH application ID:** 10207669
- **Project number:** 5R00GM129422-04
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Daniel Semlow
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $249,000
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10207669

## Citation

> US National Institutes of Health, RePORTER application 10207669, Mechanism of NEIL3-dependent ICL repair (5R00GM129422-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10207669. Licensed CC0.

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