Mechanisms of replication stress: the role of RAD52 in replication and a new vulnerability in mismatch repair deficient cancers
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Embargo End Date
2025-10-25
ICR Authors
Authors
Mocanu, C
Document Type
Thesis or Dissertation
Date
2025-04-25
Date Accepted
Abstract
Faithful duplication of chromosomes is critical for maintaining survival and preventing genomic instability.
However, both endogenous and exogenous factors can disrupt the DNA synthesis process, leading to
replication stress (RS). In turn, RS leads to the accumulation of genetic modifications causing genomic
instability, a hallmark of tumorigenesis. Understanding the causes and consequences of replication stress
is critical, not only for furthering the knowledge of the mechanistic landscape of tumorigenesis but also
for the development of better anti-cancer strategies targeting cancer vulnerabilities.
The first part of the thesis explores the consequences of RAD52 knockout in non-cancerous (RPE1) and
cancerous cells (HeLa and HCT116) under conditions of low-dose aphidicolin (APH) treatment and
translesion synthesis inhibition (TLSi). While TLSi alone reduces global DNA synthesis in cancer cells, it
has minimal impact on untransformed cells. Notably, combined TLSi and APH treatment exacerbates the
DNA synthesis defect in all cell lines, highlighting a requirement for TLS in mitigating RS. Interestingly,
RAD52 loss is synergistic with these effects only in HCT116 cells. Treatment with APH leads to underreplicated
regions that complete DNA synthesis in G2. Further, analysis of these G2 DNA synthesis
events reveals that TLS plays a critical role in maintaining replication at these regions. The data also
shows RAD52 to be necessary in HCT116, but dispensable in RPE1 cells. Investigation using chemical
inhibitors and siRNA-mediated depletion of RAD52 indicates that the phenotypes observed in HCT116
are likely due to compensatory mechanisms rather than direct functional loss of RAD52. Additionally, a
potential synthetic lethal interaction between MLH1 loss and TLSi was identified, warranting further
exploration.
The biological complexities in studying the functional role of RAD52 across various cell types and
conditions led to a shift in focus towards exploring novel synthetic lethality relationship between mismatch
repair (MMR) deficiency and TLSi in the second part of the thesis. This lethality was confirmed across
multiple untransformed cell lines (RPE1, RPE1P53KO, 1BR3, MCF10A). Notably, MMR-deficient colon
cancer cell lines exhibit increased sensitivity to TLSi compared to MMR-proficient colorectal cancers. A
proof-of-concept analysis of the TCGA cohort further demonstrates that this interaction could be exploited
in gastrointestinal and endometrial cancers. At a molecular level, it is demonstrated that combined loss
of MMR and TLS leads to ssDNA accumulation, increased p21 levels, heterochromatinization and cell
cycle delays. Furthermore, new insights reveal that loss of MMR genes alone modulates cellular
homeostasis, significantly impacting chromatin compaction, replication speed, and cell cycle progression.
Citation
2025
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Cancer and Genome Instab