Abstract
PC4 is an abundant single-strand DNA binding protein that has been implicated in transcription and DNA repair. Here, we show that PC4 is involved in the cellular DNA damage response. To elucidate the role, we used the DT40 chicken B cell model, which produces clustered DNA lesions at Ig loci via the action of activation-induced deaminase. Our results help resolve key aspects of immunoglobulin diversification and suggest an essential role of PC4 in repair pathway choice. We show that PC4 ablation in gene conversion (GC)-active cells significantly disrupts GC but has little to no effect on targeted homologous recombination. In agreement, the global double-strand break repair response, as measured by γH2AX foci analysis, is unperturbed 16 hours post irradiation. In cells with the pseudo-genes removed (GC inactive), PC4 ablation reduced the overall mutation rate while simultaneously increasing the transversion mutation ratio. By tagging the N-terminus of PC4, gene conversion and somatic hypermutation are all but abolished even when native non-tagged PC4 is present, indicating a dominant negative effect. Our data point to a very early and deterministic role for PC4 in DNA repair pathway re-routing.
Highlights
Occur to achieve the spectrum of the mutational diversity seen, considering the deamination rate being estimated at only 70–200 events/cell/day[7] and the overall creation of abasic sites is estimated at 70,000–100,000 per cell/day[8] in humans
While GC is not known to be involved in human immunoglobulin diversification, it does play a role in human genome evolution and disease where, e.g., DNA repair proteins such as BRCA1 and BRCA2 interact with the homology-searching/strand-pairing protein RAD51, and the MRE11A–RAD50–NBN (MRN) complex[10,11]
double-strand breaks (DSBs) can occur if just a few lesions in close proximity on opposite strands are repaired by long-patch BER or Nucleotide Excision Repair (NER); the resected DNA created by these pathways can lead to repair-induced DSBs13,14
Summary
Occur to achieve the spectrum of the mutational diversity seen, considering the deamination rate being estimated at only 70–200 events/cell/day[7] and the overall creation of abasic sites is estimated at 70,000–100,000 per cell/day[8] in humans. The NER-associated 3′endonuclease protein XPG is suspected of recruiting PC4 to bind ssDNA, resulting in displacement of XPG17 This could represent a mechanism to switch repair pathways in the presence of concurrent opposite strand damage, e.g. lesion clusters, and/or other factors. Mortusewicz et al.[27] showed that PC4 C-terminal-dependent foci form in response to hydroxyurea (HU) treatment, to UV-induced damage in cells pretreated with bromodeoxyuridine and to etoposide treatment, suggesting recruitment to sites of SSBs and DSBs, as well as to replication sites labeled with 5-ethynyl-2′-deoxyuridine prior to HU treatment They showed that Mre[11] inhibition and HU treatment have no effect on PC4 recruitment but do reduce RPA foci formation. As our initial observations suggested that PC4 may play a role in the radiation response (this study), as PC4 has been reported to stimulate NHEJ26, and since NHEJ-deficient DT40 cells have significantly higher levels of GC20, we tested the role of PC4 in repair of clustered lesions in light chain genes
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