Fig 2: HPF1 contributes to SSBR in CSB- and transcription-dependent manners. (A) Representative western blot showing HPF1 protein levels in CSBWT and CSBnull cells expressing a control shRNA (‘C’) and two different shRNAs targeting HPF1 (‘KD1’ and ‘KD2’). (B) Alkaline comet assays revealing the impact of HPF1 knockdown in CSBWT and CSBnull cells in the presence and absence of a-amanitin. Each circle represents the mean from 100–150 cells counted in one experiment. Shown are means ± SEM (n = 3 independent experiments). Statistical significance was determined using one-way ANOVA with Holm–Sidak's multiple comparison test. *P = 0.05, **P = 0.01, ***P = 0.001, ns: P > 0.05. (B) Representative western blot of a co-immunoprecipitation experiment revealing the effect of menadione and a-amanitin treatment on CSB–HPF1 interactions. Quantification of CSB–HPF1 co-immunoprecipitation data from two independent experiments. Shown are means ± SEM (n = 2). Statistical significance was determined by multiple unpaired t-test. **P = 0.01.

Fig 3: Model for the cooperation of CSB and PARP1/2 in SSBR at actively transcribed DNA regions. SSBs at actively transcribed DNA regions are recognized by PARP1 or PARP2, which then recruit CSB. CSB recruitment does not require ATP hydrolysis by CSB or the PARylation activity of PARP1/2. Once bound, CSB facilitates the recruitment or stabilizes the association of HPF1 with oxidized chromatin, leading to the alteration of the PARP1/2 substrate specificity to include histone serine PARylation. Chromatin-bound CSB also facilitates the recruitment of the SSBR machinery, such as XRCC1 and DNA ligase 3. Execution of SSBR requires the ATP-dependent chromatin remodeling activity of CSB, which may be used to facilitate PARP1/2 dissociation and/or alter chromatin structure at actively transcribed DNA regions to promote efficient repair.