BRCA1 is an important mediator of the DNA damage response, which promotes homologous recombination (HR) and antagonizes 53BP1-dependent non-homologous end joining in S/G2 phase. But how this is achieved remains unclear. Here, we report that the E3 ubiquitin ligase UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) directly participates in the interplay between BRCA1 and 53BP1. Mechanistically, UHRF1 is recruited to DNA double-strand breaks (DSBs) by BRCA1 in S phase, which requires the BRCT domain of BRCA1 and phosphorylated Ser674 of UHRF1. Subsequently, UHRF1 mediates K63-linked polyubiquitination of RIF1, and results in its dissociation from 53BP1 and DSBs thereby facilitating HR initiation. Thus, UHRF1 is a key regulator of DSB repair choice, which is separate from its role in heterochromatin formation and epigenetic regulator.
Pif1 DNA helicase is a potent unwinder of G-quadruplex (G4) structures in vitro and functions to maintain genome stability at G4 sequences in Saccharomyces cerevisiae. Here, we developed and utilized a live-cell imaging approach to quantitatively measure the progression rates of single replication forks through different G4 containing sequences in individual yeast cells. We show that in the absence of Pif1, replication rates through specific lagging strand G4 sequences in vivo is significantly decreased. In contrast, we found that in the absence of Pif1, replication rates through the same G4s on the leading strand are not decreased relative to the respective WT strains, showing that Pif1 is essential only for efficient replication through lagging strand G4s. Additionally, we show that a canonical PIP sequence in Pif1 interacts with PCNA and that replication through G4 structures is significantly slower in the absence of this interaction in vitro and in vivo. Thus, Pif1–PCNA interaction is essential for optimal replisome progression through G4 sequences, highlighting the importance of coupling between Pif1 activity and replisome progression during yeast genome replication.
G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5′ to 3′ helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5′-3′-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3′-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.
Background: Proper regulation of DSB resection is key to genome maintenance. Results: 14-3-3s bind to Exo1 and restrain its damage association and resection activity by counteracting the function of PCNA. Conclusion: Exo1 activity is controlled by both positive and negative regulators to ensure a proper level of DNA end resection. Significance: Our data reveal a key mechanism that controls the DNA end resection process.
Exonuclease 1 (Exo1) has important roles in DNA metabolic transactions that are essential for genome maintenance, telomere regulation and cancer suppression. However, the mechanisms for regulating Exo1 activity in these processes remain incompletely understood. Here we report that Exo1 activity is regulated by a direct interaction with poly(ADP-ribose) (PAR), a prominent posttranslational modification at the sites of DNA damage. This PAR-binding activity promotes the early recruitment of Exo1 to sites of DNA damage, where it is retained through an interaction with PCNA, which interacts with the C-terminus of Exo1. The effects of both PAR and PCNA on Exo1 damage association are antagonized by the 14-3-3 adaptor proteins, which interact with the central domain of Exo1. Although PAR binding inhibits both the exonuclease activity and the 5’ flap endonuclease activity of purified Exo1, the pharmacological blockade of PAR synthesis does not overtly affect DNA double-strand break end resection in a cell free Xenopus egg extract. Thus, the counteracting effects of PAR on Exo1 recruitment and enzymatic activity may enable appropriate resection of DNA ends while preventing unscheduled or improper processing of DNA breaks in cells.
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