# DNA damage signaling to dormant origins of replication

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2022 · $350,834

## Abstract

Higher eukaryotes evolved with mechanisms that initiate DNA replication at multiple origins on multiple
chromosomes. Activation of the replicative helicase at a single origin in each of ~50,000 replicons is sufficient
to replicate the human genome in the absence of stress. These ~50,000 origins are selected from a five- to
tenfold excess of licensed origins. Activation of additional replicative helicases at origins that would otherwise
be passively replicated is observed after stress. This plasticity in origin use is a simple mechanism to recover
DNA replication between stalled and collapsed replication forks. The mechanism(s) that limits origin firing to one
per replicon in the absence of stress is not known. We recently reported that the DNA damage signaling kinases
ATR and Chk1 inhibit activation of the replicative helicase in the absence of stress. In preliminary studies, we
show that Chk1 kinase activity is strictly associated with ATR kinase-dependent phosphorylations on Chk1 and
that these have an astonishingly short half-life in cells. We propose that this is a highly innovative mechanism
that localizes Chk1 kinase activity to the immediate vicinity of ATR at active replicative helicases. We also show
that Rif1, which has been implicated in the regulation of replication timing previously, is phosphorylated and that
phosphorylated Rif1 binds protein phosphatase 1α (PP1α). Based upon these findings, we hypothesize that
Chk1 kinase activity generates a ring of Rif1-PP1α around active replicative helicases and that this limits Cdc7
kinase-dependent origin firing across a replicon. In Aim 1 we will investigate a new mechanistic paradigm for
localizing DNA damage signaling to a small volume of the nucleus in the absence of stress. In Aim 2 we will
investigate the molecular mechanism that limits activation of the replicative helicase across a replicon in the
absence of stress. In Aim 3 we will investigate the impact of ATR kinase inhibition and conformal radiation on
immune responses in tumor bearing mice. These studies are highly impactful as they will identify a fundamental
mechanism that determines inter-origin distance, genome stability and the rate of cell division in higher
eukaryotes. Since this mechanism may be attenuated in T cells, these studies will provide fundamental insights
into adaptive immune responses. Our studies may have an immediate impact as the ATR and Chk1 kinase
inhibitors used here are in clinical trials.

## Key facts

- **NIH application ID:** 10295771
- **Project number:** 5R01CA236367-03
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** CHRISTOPHER J. BAKKENIST
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $350,834
- **Award type:** 5
- **Project period:** 2019-12-01 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10295771

## Citation

> US National Institutes of Health, RePORTER application 10295771, DNA damage signaling to dormant origins of replication (5R01CA236367-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10295771. Licensed CC0.

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