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The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.

In metazoans, the largest sirtuin, SIRT1, is a nuclear protein implicated in epigenetic modifications, circadian signaling, DNA recombination, replication, and repair. Our previous studies have demonstrated that SIRT1 binds replication origins and inhibits replication initiation from a group of potential initiation sites (dormant origins). We studied the effects of aging and SIRT1 activity on replication origin usage and the incidence of transcription–replication collisions (creating R-loop structures) in adult human cells obtained at different time points during chronological aging and in cancer cells. In primary, untransformed cells, SIRT1 activity declined and the prevalence of R-loops rose with chronological aging. Both the reduction in SIRT1 activity and the increased abundance of R-loops were also observed during the passage of primary cells in culture. All cells, regardless of donor age or transformation status, reacted to the short-term, acute chemical inhibition of SIRT1 with the activation of excessive replication initiation events coincident with an increased prevalence of R-loops. However, cancer cells activated dormant replication origins, genome-wide, during long-term proliferation with mutated or depleted SIRT1, whereas, in primary cells, the aging-associated SIRT1-mediated activation of dormant origins was restricted to rDNA loci. These observations suggest that chronological aging and the associated decline in SIRT1 activity relax the regulatory networks that protect cells against excess replication and that the mechanisms protecting from replication–transcription collisions at the rDNA loci manifest as differentially enhanced sensitivities to SIRT1 decline and chronological aging. [ABSTRACT FROM AUTHOR]

The cell cycle is modulated by ubiquitin ligases, including CRL4, which facilitate degradation of the chromatin-bound substrates involved in DNA replication and chromosome segregation. One of the members of the CRL4 complex, RepID (DCAF14/PHIP), recognizes kinetochore-localizing BUB3, known as the CRL4 substrate, and recruits CRL4 to the chromatin/chromosome using the WD40 domain. Here, we show that the RepID WD40 domain provides different platforms to CRL4 and BUB3. Deletion of the H-box or exon 8 located in the RepID WD40 domain compromises the interaction between RepID and CRL4, whereas BUB3 interacts with the exon 1–2 region. Moreover, deletion mutants of other exons in the WD40 domain lost chromatin binding affinity. Structure prediction revealed that the RepID WD40 domain has two beta-propeller folds, linked by loops, which are possibly crucial for chromatin binding. These findings provide mechanistic insights into the space occupancy of the RepID WD40 domain to form a complex with CRL4, BUB3, or chromatin. • RepID WD40 domain is crucial for binding to chromatin and proteins. • WD40 domain of RepID forms a two-β propeller configuration connected by a loop. • RepID WD40 domain provides different platforms for proteins and chromatin. [ABSTRACT FROM AUTHOR]

Cells activate distinctive regulatory pathways that prevent excessive initiation of DNA replication to achieve timely and accurate genome duplication. Excess DNA synthesis is constrained by protein–DNA interactions that inhibit initiation at dormant origins. In parallel, specific modifications of pre-replication complexes prohibit post-replicative origin relicensing. Replication stress ensues when the controls that prevent excess replication are missing in cancer cells, which often harbor extrachromosomal DNA that can be further amplified by recombination-mediated processes to generate chromosomal translocations. The genomic instability that accompanies excess replication origin activation can provide a promising target for therapeutic intervention. Here we review molecular pathways that modulate replication origin dormancy, prevent excess origin activation, and detect, encapsulate, and eliminate persistent excess DNA. Cells have evolved specialized mechanisms to avoid the deleterious effects of excess initiation from replication origins. During unperturbed proliferation, most potential replication origins are dormant and do not initiate replication but are prepared for activation to facilitate complete genome duplication if cells encounter replication stress. Cells prevent initiation at dormant origins by adjusting chromatin structure to constrain the assembly and activation of pre-replication complexes, targeting components of the replication machinery for degradation or modulating specific protein–DNA interactions at origins. Cancer cells often lack the controls that prevent excess activation of replication origins, resulting in the amplification of extrachromosomal DNA that can persist if they contain genes that can confer a selective growth advantage. Cells activate metabolic and inflammatory responses to sense and encapsulate and eliminate excess DNA. Because excess replication origin activation can directly or indirectly lead to genomic instability, targeting the pathways that prevent excess replication or eliminate excess DNA is a promising avenue for therapeutic intervention. [ABSTRACT FROM AUTHOR]