KBM-mediated interactions with KU80 promote cellular resistance to DNA replication stress in CHO cells.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

DNA combing versus DNA spreading and the separation of sister chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example, in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves sister chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading typically does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

DNA Combing versus DNA Spreading and the Separation of Sister Chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves single chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair.

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1-/- mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1-/- cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.