Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is largely inactivated in mitosis. Instead, mitotic cells rely on alternative repair processes such as microhomology-mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). How these mitotic DNA repair pathways are functionally regulated remains unclear. Here we reveal that the CIP2A-TOPBP1 complex plays an essential regulatory role by facilitating the mitotic recruitment of both SMX complex components and Polθ to mitotic chromatin. Recruitment of the SMX complex components is driven by CDK1-dependent phosphorylation of SLX4 at Thr1260, enabling its interaction with TOPBP1 BRCT domains 1/2, thereby promoting MiDAS. Concurrently, CIP2A promotes efficient mitotic localisation of Polθ to facilitate MMEJ. The simultaneous functional disruption of both MiDAS and MMEJ pathways upon CIP2A loss provides rationale for the synthetic lethality observed in BRCA1 or 2-deficient cells. These findings position the CIP2A-TOPBP1 axis as a central regulatory hub for mitotic DNA repair, highlighting therapeutic opportunities in tumours characterised by HR deficiency or elevated replication stress.

Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is inactivated in mitosis. Mitotic cells instead rely on the mechanisms of microhomology mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). However, how these pathways are regulated in mitosis remains unknown. Here we reveal the CIP2A-TOPBP1 complex facilitates recruitment of SMX complex components to mitotic chromatin marked by CIP2A, through a CDK1-dependent interaction between TOPBP1 BRCT 1/2 and SLX4 phospho-threonine1260, that drives MiDAS. Furthermore, CIP2A promotes the recruitment of Polθ to facilitate mitotic MMEJ. This defines a mechanistic framework for mitotic DSB repair, where simultaneous disruption of MiDAS and MMEJ pathways underpins the synthetic lethality observed in BRCA1/2-deficient cells following CIP2A depletion. These findings provide critical insights into mitotic DNA repair and highlights therapeutic opportunities in HR deficient tumours.

The preservation and faithful propagation of genetic information is essential for all life forms and depends on cellular pathways that enable replication, recombination, and repair of DNA. The multifunctional XPF-ERCC1 DNA endonuclease complex acts in several DNA repair pathways and interacts with numerous partner proteins and large DNA repair assemblies, including the nucleotide excision repair machinery and the SMX tri-endonuclease complex. Here, we report structures of XPF-ERCC1 in complex with the DNA repair factors SLX4 and SLX4IP, thereby identifying key residues responsible for direct interactions with XPF-ERCC1. When introduced into human cells, point mutations in these interfaces impair the interactions between XPF-ERCC1 and SLX4 or SLX4IP, and disruption of the XPF-SLX4IP interface leads to cis-platin sensitivity. Furthermore, our data reveal the structure of the human XPF-ERCC1-SLX4IP-SLX4 ^330-555 complex with DNA bound at its active site, and they complete the structural characterisation of molecular interactions required to assemble the SMX complex.

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress and protect stalled replication forks. All such responses rely on the formation of Replication Protein A (RPA)-ssDNA complexes, yet supra-physiological binding of RPA to ssDNA is toxic. How cells regulate RPA availability to promote fork protection and genome stability is largely unknown. Here we establish that during replication excess RPA is sequestered by monomeric actin and released upon replicative stress through transition to polymeric actin state. Impairment in actin nucleation leads to RPA sequestration, deprotection of ssDNA generated at the stressed forks and consequently, catastrophic fork collapse and hypersensitivity to replication inhibitors. In line with this, we show that increasing RPA load is sufficient to restore efficient fork protection in actin polymerization mutants. Collectively, this work identifies a simple yet robust RPA-buffering mechanism regulating its availability to bind ssDNA and protect replication forks against nucleolytic degradation. Inhibition of this pathway could be of therapeutic interest in treatment of cancers.