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Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells

ABSTRACT: Attaching/effacing(A/E) pathogens induce DNA damage and colorectal cancer by injecting effector proteins into host cells via the type III secretion system (T3SS). EspF is one of the T3SS-dependent effector proteins exclusive to A/E pathogens, which include enterohemorrhagicEscherichia coli(EHEC). The role of EspF in the induction of double-strand breaks (DSBs) and the phosphorylation of the repair protein SMC1 has been demonstrated previously. However, the process of damage accumulation and DSB formation has remained enigmatic, and the damage response is not well understood. Here, we first showed a compensatory increase in the mismatch repair (MMR) proteins MutS homolog 2 (MSH2) and MSH6, as well as poly(ADP-ribose) polymerase 1 (PARP1), followed by a dramatic decrease, threatening cell survival in the presence of EspF. Flow cytometry revealed that EspF arrested the cell cycle at the G2/M phase to facilitate DNA repair. Subsequently, 8-oxoguanine (8-oxoG) lesions, a marker of oxidative damage, were assayed by ELISA and immunofluorescence, which revealed the accumulation of 8-oxoG from the cytosol to the nucleus. Furthermore, the status of single-stranded DNA (ssDNA) and DSBs was confirmed. We observed that EspF accelerated the course of DNA lesions, including 8-oxoG and unrepaired ssDNA, which were converted into DSBs; this was accompanied by the phosphorylation of Replication protein A 32 (RPA32) in repair-defective cells. Collectively, these findings reveal that EspF triggers various types of oxidative DNA lesions with impairment of the DNA damage response and may result in genomic instability and cell death, offering novel insight into the tumorigenic potential of EspF.

ORGANISM(S): Homo sapiens

PROVIDER: GSE255129 | GEO | 2024/02/14

REPOSITORIES: GEO

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The E3 ubiquitin ligase RFWD3 is required for translesion DNA synthesis (total proteome)

Project description:Lesions on DNA can uncouple DNA synthesis from helicase unwinding, generating stretches of unreplicated single stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication and thereby avoid the generation of DNA double-stranded breaks (DSBs), a major source of genomic instability. Gap filling repair involves either translesion DNA synthesis (TLS) or template switching (TS) to bypass the lesion. At the heart of these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes non-specific ubiquitylation of proteins on ssDNA, to enhance protein accumulation and stimulate gap filling repair. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin, including ubiquitin and proteins known to ubiquitylate and interact with ubiquitylated PCNA. As a result, PCNA ubiquitylation is severely inhibited without RFWD3, and TLS across different DNA lesions drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it triggers non-specific ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

2020-12-15 | PXD021445 | Pride

The E3 ubiquitin ligase RFWD3 is required for translesion DNA synthesis

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2020-12-15 | PXD018217 | Pride

Genome-wide mapping of early replication fragile sites (ERFS)

Project description:DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins RPA, SMC5, gamma-H2AX, and BRCA1 in B cells subjected to replication stress. Protein-DNA association for four DNA damage response proteins (RPA, SMC5, g-H2AX, BRCA1), BrdU incorporation, and gene transcription in B lymphocytes with and without hydroxyurea treatment were examined.

2013-02-04 | E-GEOD-43504 | biostudies-arrayexpress

HDAC1 modulates OGG1-initiated oxidative DNA damage repair, brain aging, and Alzheimer’s disease pathology

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2020-03-30 | GSE115437 | GEO

Genome-wide mapping of early replication fragile sites (ERFS)

2013-02-04 | GSE43504 | GEO

RPA accumulation during class switch recombination represents 5'-3' DNA end resection during the S-G2/M phase of the cell cycle

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2013-01-02 | E-GEOD-42451 | biostudies-arrayexpress

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2022-06-24 | GSE188779 | GEO

Strand-specific ChIP-seq at DNA breaks distinguishes single versus doubled stranded DNA binding and refutes single stranded nucleosomes

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2021-03-01 | GSE149807 | GEO

RPA accumulation during class switch recombination represents 5'-3' DNA end resection during the S-G2/M phase of the cell cycle

2013-01-02 | GSE42451 | GEO

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