A double-ring of human RAD52 remodels replication forks restricting fork reversal

Honda, Masayoshi; Razzaghi, Mortezaali; Gaur, Paras; Malacaria, Eva; Marozzi, Giorgia; Biagi, Ludovica Di; Aiello, Francesca Antonella; Paintsil, Emeleeta A.; Stanfield, Andrew J.; Deppe, Bailey J.; Gakhar, Lokesh; Schnicker, Nicholas J.; Ashley Spies, M.; Pichierri, Pietro; Spies, Maria

doi:10.1101/2023.11.14.566657

Cited by 4 publications

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

An integrative structural study of the human full-length RAD52 at 2.2 Å resolution

Balboni,

Marotta,

Rinaldi

et al. 2024

Commun Biol

View full text Add to dashboard Cite

Human RAD52 (RAD52) is a DNA-binding protein involved in many DNA repair mechanisms and genomic stability maintenance. In the last few years, this protein was discovered to be a promising novel pharmacological target for anticancer strategies. Although the interest in RAD52 has exponentially grown in the previous decade, most information about its structure and mechanism still needs to be elucidated. Here, we report the 2.2 Å resolution cryo-EM reconstruction of the full-length RAD52 (FL-RAD52) protein. This allows us to describe the hydration shell of the N-terminal region of FL-RAD52, which is structured in an undecamer ring. Water molecules coordinate with protein residues to promote stabilization inside and among the protomers and within the inner DNA binding cleft to drive protein-DNA recognition. Additionally, through a multidisciplinary approach involving SEC-SAXS and computational methods, we comprehensively describe the highly flexible and dynamic organization of the C-terminal portion of FL-RAD52. This work discloses unprecedented structural details on the FL-RAD52, which will be critical for characterizing its mechanism of action and inhibitor development, particularly in the context of novel approaches to synthetic lethality and anticancer drug discovery.

show abstract

An integrative structural study of the human full-length RAD52 at 2.2 Å resolution

Balboni,

Marotta,

Rinaldi

et al. 2024

Commun Biol

View full text Add to dashboard Cite

show abstract

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single-stranded DNA gaps

Pale,

Khatib,

Nusawardhana

et al. 2024

Nucleic Acids Research

View full text Add to dashboard Cite

Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is, however, imperfect and continuously evolving. Recent advances highlighted single-stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon restart of DNA synthesis downstream of cisplatin-induced lesions through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wild-type cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway. RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing and cisplatin sensitization.

show abstract

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single stranded DNA gaps

Pale,

Khatib,

Nicolae

et al. 2024

Preprint

View full text Add to dashboard Cite

Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is however imperfect, and continuously evolving. Recent advances in the field highlighted single stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon the arrest of replication forks at sites of cisplatin adducts, and restart of DNA synthesis downstream of the lesion through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wildtype cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway, and show that RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing, shedding light on the mechanisms of cisplatin sensitization in cancer therapy.

show abstract

A double-ring of human RAD52 remodels replication forks restricting fork reversal

Cited by 4 publications

References 67 publications

An integrative structural study of the human full-length RAD52 at 2.2 Å resolution

An integrative structural study of the human full-length RAD52 at 2.2 Å resolution

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single-stranded DNA gaps

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single stranded DNA gaps

Contact Info

Product

Resources

About