# Mechanism and Regulation of DNA recombination in Saccharomyces cerevisiae

> **NIH NIH R01** · BAYLOR COLLEGE OF MEDICINE · 2020 · $306,472

## Abstract

Homologous recombination (HR) plays an essential role in maintaining stability of genetic information, and
even a minor deficiency in HR leads to severe diseases including cancer. Recombination repairs DNA double-
strand breaks (DSBs) that occur spontaneously, or are induced by chemicals or irradiation such as used in
cancer therapy. Nearly all we know about recombination processes comes from studies of two-ended double
strand breaks (DSBs) induced by endonucleases (e.g. I-SceI, HO). However, it is well established that
spontaneous chromosomal breaks are predominantly single-ended DSBs (seDSBs), as they arise during DNA
replication when a replication fork runs into a nick. In bacteria that contain a single replication origin per
genome, broken forks are repaired by Pri proteins capable of reloading the replisome at any genomic location.
However, Pri proteins are not conserved, and the mechanism of broken fork repair in eukaryotes remains
undefined. Our long-term goal is to understand the molecular mechanisms and regulation of DSB repair
including broken replication fork repair, and to understand how deficiencies in these processes affect genomic
instability. The objective of this project is to define the mechanistic features of Broken Fork Repair (BFR),
which is the most common, yet poorly understood, type of DSB repair. We propose that eukaryotes repair
broken replication forks using a combination of the structure-specific nuclease Mus81/Mms4 and a converging
fork initiated at the next active or damage-activated origin. We further propose that this mechanism restricts the
usage of highly mutagenic DNA synthesis via the well-characterized Break Induced Replication (BIR) process.
The central question is whether eukaryotes are able to reestablish replication forks at the site of fork breakage
as demonstrated in bacteria. What are the genetic requirements for broken fork repair and how do they differ
from mutagenic BIR? What is the fate of replisome proteins at broken forks? These questions will be
addressed in the yeast model organism Saccharomyces cerevisiae, where all replication origins are annotated
and Flp recombinase-induced broken fork assays are available. We will define whether functional forks can be
reestablished and whether dormant origins are activated in the vicinity of the broken fork using the hydrolytic
end sequencing (HydEn-seq) method. The stability of the replisome after fork breakage will be studied using
chromatin immunoprecipitation. We will also study the role and regulation of structure-specific nucleases in the
repair of broken forks. The most common types of genomic rearrangements that occur during BFR and BIR
stem from template switches and half crossovers. We will identify the genetic requirements for these events. At
the conclusion of this project we expect to: (i) provide new molecular tools to study BFR, (ii) delineate the
major mechanism of BFR, and (iii) uncover mechanisms that prevent mutagenic BIR, which is belie...

## Key facts

- **NIH application ID:** 9912782
- **Project number:** 5R01GM080600-13
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** Grzegorz A Ira
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $306,472
- **Award type:** 5
- **Project period:** 2007-05-01 → 2021-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9912782

## Citation

> US National Institutes of Health, RePORTER application 9912782, Mechanism and Regulation of DNA recombination in Saccharomyces cerevisiae (5R01GM080600-13). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/9912782. Licensed CC0.

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