It is of interest to
It is of interest to consider an explanation for the apparent phosphorylation of hSSB1 in undamaged cells. One such may be that in order to execute a rapid response to replication inhibition, basal S134 phosphorylation could function to keep hSSB1 in a ‘primed’ state prior to stress. Such an arrangement may be consistent with our previous description of hSSB1 as an early responder to DNA damage. This is demonstrated by the rapid co-localisation of hSSB1 with sites of stalled replication in response to hydroxyurea treatment and its subsequent roles in ATR recruitment . A similar ‘priming’ approach has also been suggested for Chk1, where phosphorylation of S317 and S345 is maintained under a suppressive state by PP1 during un-perturbed cell cycles to allow for rapid checkpoint activation in response to replication fork stalling . As basal hSSB1 phosphorylation may involve DNA-PK function, this scenario suggests that a pool of activated DNA-PK must also exist in un-exogenously damaged cells. This is supported by the similar increase in DNA-PK auto-phosphorylation that has been observed following okadaic Sunitinib Malate australia treatment of cells , or depletion of PP5 . It is worth noting, however, that basal phosphorylation of RPA32 S33 was also observed in cells, which increased at a similar rate to hSSB1 S134 phosphorylation following hydroxyurea treatment. RPA32 S33 phosphorylation in undamaged cells has previously been found to occur in the late S- and G2 phases of the cell cycle and has been attributed to the persistence of endogenously stalled replication forks . A similar regulatory mechanism may thereby also explain the detection of S134 phosphorylated hSSB1 in undamaged cells. As detection of S134 phosphorylation predominantly required transient overexpression of hSSB1, we cannot however rule out that the high degree of basal hSSB1 phosphorylation observed was due the equilibrium between S134 phosphorylation and de-phosphorylation being somewhat altered from endogenous ratios.
Whilst our studies of WT and S134E hSSB1 DNA-binding did not yield any additional insight into the molecular function of S134 phosphorylation, we were surprised to find that hSSB1 can bind fork substrates, especially given the negligible interaction of hSSB1 with dsDNA duplexes reported previously . Interestingly, a similar observation was recently reported for the annealing helicase SMARCAL1, a protein that also has minimal affinity for dsDNA, although which readily binds model replication forks lacking exposed ssDNA . In this work, the authors suggested SMARCAL1 might capture small amounts of ssDNA that are exposed due to ‘breathing’ of the dsDNA regions adjacent to the fork junction. It is tempting to consider that hSSB1 may bind this structure through a similar means. Indeed, these results are somewhat reminiscent of our finding that hSSB1 is able to bind a dsDNA substrate containing a single 8-oxoguanine modification, potentially due to a similar localised de-stabilisation of the DNA duplex . Although we have previously demonstrated that hSSB1 localises to replication forks following their disruption , the mechanistic consequence of fork-junction binding however remains unclear. These data nevertheless suggest that S134 phosphorylation is unlikely to alter hSSB1 DNA-binding and may instead alter hSSB1 via an alternative means, such as by modulating the interaction with an as yet unidentified protein-binding partner.
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Introduction Induction of DNA double stranded breaks (DSBs) by radiation is considered an effective approach for the treatment of cancer (Jeggo and Lobrich, 2007, McKinnon and Caldecott, 2007). Both radiation and a wide range of anticancer agents act by the induction of DSB in cancer cells, thereby causing cell death via various death pathways. (Ghiassi-Nejad et al., 2002, Desai et al., 2005). The direct impact of ionizing radiation (IR) on the double helix structure of DNA introduces DNA lesions in the form of DSBs and is often used as an adjunct to chemotherapy (Brenner and Ward, 1992, Takahashi and Ohnishi, 2005). To maintain genomic integrity and prevent chromosomal rearrangements, cells are programmed to repair DSBs through numerous biological pathways (Bennett, Lewis, Baldwin, & Resnick, 1993). The two well-known pathways through which DSBs can be repaired are non-homologous DNA end joining (NHEJ) and homologous recombination (HR). NHEJ is a repair process where the broken DNA ends are directly joined/ligated together without following any specific homology sequencing whereas HR requires an identical sequence for repair of DSBs(Neal & Meek, 2011). The presence of DSBs initiates the activation and binding of the heterodimer complex Ku80 (Ku70 and Ku86) to the broken DNA strands (Mimori et al., 1981, Paillard and Strauss, 1991, Falzon et al., 1993, Walker et al., 2001). This bonding of Ku to DSBs initiates the recruitment of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) forming a Ku 70/80 heterodimer and binding the DNA-PKcs holoenzyme to the DSBs. Once the two broken ends of DSBs bind with the Ku70/80-DNAPKcs holoenzyme various processing factors such as Artemis, Polynucleotide kinase phosphatase (PNKP), terminal deoxynucleotidyltransferase (Tdt), and polymerases stimulate the end processing of the non ligatable DNA ends (Neal & Meek, 2011). Following end processing, DSB ends are appropriately ligated together with the help of the XRCC4-LigIV ligase complex thus completing DSB repair by NHEJ (Ma et al., 2004, Dueva and Iliakis, 2013). DNA-PKcs has been grouped under the family of phosphatidylinositol 3-kinase-related kinases (PIKK) and is one of the largest kinases (465 kDa) to be completely involved in DNA-DSB repair by NHEJ (Lempiainen & Halazonetis, 2009). Evidence suggests that up-regulation and induction of DNA-PKcs and Ku following radiation leads to selective radiation resistance in recurrent tumours (Shintani et al., 2003, Beskow et al., 2009). Previous studies have demonstrated strategies to target DNA-PKcs in an attempt to improve radiotherapy. These have included the use of ATP competitive small molecule inhibitors of DNA-PKcs such as vanillin, NU7026, NU7441, IC87361 and SU11752 (Durant and Karran, 2003, Hollick et al., 2003, Ismail et al., 2004, Leahy et al., 2004, Gene et al., 2005). All these compounds sensitized human cell models to radiation without significant cellular toxicity (Durant and Karran, 2003, Kashishian et al., 2003, Ismail et al., 2004, Gene et al., 2005, Nutley et al., 2005, Zhao et al., 2006, Shaheen et al., 2011). Notable was the DNA-PKcs IC50 of NU7441 of 14 nM in a non-cellular enzyme based assay system which makes this the most potent inhibitor identified so far with no inhibition against other related kinases such as ATM and ATR (Leahy et al., 2004).