CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin

Abstract

Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81–RAD52–POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ–LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2.

CSB and SMARCAL1 compete for RPA32 at stalled forks and differentially control the fate of stalled forks in BRCA2-deficient cells

“…To investigate whether PRIMPOL mediates unrestrained fork progression in CSB-KO cells, we knocked down PRIMPOL in both U2OS WT and CSB-KO cells (Figure 6C ). DNA fiber analysis revealed that depletion of PRIMPOL abrogated the restoration of the ratio of CldU/IdU in CSB-KO cells in the presence of 50 μM HU (Figure 6D and E ), in agreement with our previous finding that PRIMPOL mediates unrestrained fork progression in CSB-KO cells in response to a low dose of camptothecin (CPT) ( 36 ). PRIMPOL-mediated fork repriming is associated with accumulation of ssDNA gaps ( 64–66 ).…”

“…S1 nuclease assays were done as described ( 36 ). To detect ssDNA gaps, cells were first incubated with 25 μM IdU for 20 min and then 250 μM CldU for 60 min in the presence of 50 μM HU.…”

“…Like SMARCAL1, CSB (Cockayne syndrome group B) is a DNA translocase that belongs to the SNF2 helicase family ( 12 ). First reported for its role in transcription-coupled nucleotide excision repair (TC-NER) ( 28 , 29 ), CSB has been implicated in DNA DSB repair ( 30–34 ) and the replication stress response ( 17 , 35 , 36 ). CSB possesses an intrinsic fork reversal activity in vitro ( 17 ), which is likely to be highly regulated in vivo .…”