ATR and ATM-Dependent Movement of BLM Helicase during Replication Stress Ensures Optimal ATM Activation and 53BP1 Focus Formation

Abstract

The BLM helicase, a deficiency that markedly increases cancer incidence in humans, is required for optimal repair during DNA replication. We show that BLM rapidly moves from PML nuclear bodies to damaged replication forks, returning to PML bodies several hours later, owing to activities of the DNA damage response kinases ATR and ATM, respectively. Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different sub-cellular compartments after replication stress. Unexpectedly, fibroblasts lacking BLM were deficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failed to form foci following replication stress. Expression of a dominant p53 mutant or helicase-deficient BLM restored replication stress-induced 53BP1 foci, but only mutant p53 restored optimal ATM activation. Thus, optimal repair of damaged replication fork lesions likely requires both ATR and ATM. BLM recruits 53BP1 to these lesions independent of its helicase activity, and optimal activation of ATM requires both p53 and BLM helicase activities.

Phosphorylation of BLM, Dissociation from Topoisomerase IIIα, and Colocalization with γ-H2AX after Topoisomerase I-Induced Replication Damage

“…Thus, under replicative stress, BLM might act as a transducer facilitating H2AX phosphorylation in response to replication damage. This proposed role for BLM is consistent with a recent report showing that cells lacking BLM are deficient in activating/phosphorylating the DNA damage sensor kinase ATM at serine 1981 in response to hydroxyurea (14). The absence of BLM has also been shown to impair the focus forming ability of the MRN complex and BRCA1 in response to hydroxyurea (13,21).…”

The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability

“…Furthermore, we have observed a slight decrease in the number of RECQL4-and PML-colocalizing foci after treatment of cells with the DSB-inducing agent etoposide. Similarly to this observation, it has recently been reported that, BLM helicase relocalizes from PML NBs after replication stall due to HU treatment (Davalos et al, 2004). Thus, our results also suggest that RECQL4 leaves PML foci and, after induction of DNA damage, relocalizes in a similar manner as BLM.…”

p53 in recombination and repair

“…A recent study also showed that Blm itself is required for optimal activation of Atm, the kinase which regulates DSB repair proteins and induces checkpoint signalling. 145 Evidence for a Regulatory Role of p53 in NHEJ With respect to a possible involvement of p53 in NHEJ, results that initially appeared contradictory were reported. Thus, two groups observed an increase in rejoining of linearised plasmid DNA in the presence of wild-type or temperature-sensitive mutant p53.…”

“…This scenario is especially interesting, since Blm also recruits 53Bp1 to replication fork lesions and facilitates physical interaction between p53 and p53 binding protein 1 (53Bp1), a recruiting factor for Atm targeting to DSBs. 144,145 Blm-p53 interactions most likely promote phosphorylation of p53 through Atm and Atr, which in turn may p53 in recombination and repair SA Gatz and L Wiesmüller stabilise complexes with other DSB repair proteins like Rad51, Rad54, and RPA. A recent study also showed that Blm itself is required for optimal activation of Atm, the kinase which regulates DSB repair proteins and induces checkpoint signalling.…”