# Regulation of replication and recombination intermediates

> **NIH NIH R01** · SLOAN-KETTERING INST CAN RESEARCH · 2022 · $100,465

## Abstract

Faithful duplication of the genome requires regulation of replication forks that stall at numerous template
blockages. Failure to assist stalled replication forks can lead to incomplete replication and many types of genetic
alterations underlying DNA fragility syndromes and tumorigensis. Non-histone proteins tightly bound to DNA
(protein barriers) are a major cause of fork blockade, and a large portion of these are located inside the repetitive
ribosomal DNA (rDNA). rDNA organizes nucleoli and constitutes 10-30% of the genome across species. As such,
rDNA replication influences overall genomic stability as well as RNA and protein synthesis. rDNA protein barriers
have unique features such as greater topological stress due to high levels of rRNA transcription and requirement
of extended maintenance of the replisome. Mechanisms that can ensure rDNA replication completion given these
challenges are unclear. Excitingly, our recent data in yeast suggest that the conserved eight-subunit Smc5/6
complex provides an integrated solution for coping with unique challenges at rDNA. We found that Smc5/6 is
essential for completing replication at rDNA but not at non-rDNA regions. We further determined that Smc5/6
limits replication fork reversal at rDNA protein barriers. Our new data let us propose that Smc5/6 uses the
combined activities of its subunits to regulate stalled forks at rDNA protein barriers and ensure proper rDNA
replication termination. We plan to test this central hypothesis using a combination of molecular, genetic, and
biochemical approaches in Aim 1.
 When stalled replication forks fail to recover, collapsed forks and unreplicated DNA gaps can be repaired
by homologous recombination, generating recombination intermediates such as Holliday junctions. Promptly
resolving these structures is critical for preventing DNA entanglement during mitosis, which can lead to anaphase
bridges, micronuclei formation, and genomic instability. Studies from us and others have uncovered multiple
regulatory factors that are critical for Holliday junction removal. However, their functional mechanisms remain to
be elucidated. Our current research on one of the conserved regulatory factors, the Esc2 protein, which is critical
for genomic stability, leads to new models for its functional mechanisms. In particular, we suggest that Esc2 uses
a bimodal strategy for enhancing HJ dissolution, including both a structural contribution and a SUMO-mediated
mechanism. In Aim 2, we plan to test this model and define how HJ clearance is enabled by Esc2. To accomplish
the goals in this proposal, we will use high-resolution assays in the highly effective yeast system. Outcomes of
this proposed work will expand our view of several processes, including how rDNA replication completion is
achieved, how replication fork is regulated in a context-specific manner, and how recombination intermediate
removal can be assisted by regulatory proteins. As these processes are intimately linked to...

## Key facts

- **NIH application ID:** 10689591
- **Project number:** 3R01GM131058-04S1
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** Xiaolan Zhao
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $100,465
- **Award type:** 3
- **Project period:** 2019-08-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10689591

## Citation

> US National Institutes of Health, RePORTER application 10689591, Regulation of replication and recombination intermediates (3R01GM131058-04S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10689591. Licensed CC0.

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