Mitotic phosphorylation of Bloom helicase at Thr182 is required for its proteasomal degradation and maintenance of chromosomal stability

Abstract

Mutations in Bloom helicase (BLM) lead to Bloom Syndrome (BS). BS is characterized by multiple clinical manifestations including predisposition to a wide spectrum of cancers. Studies have revealed the mechanism of BLM recruitment after stalled replication and its role during the repair of DNA damage. We now provide evidence that BLM undergoes K48-linked ubiquitylation and subsequent degradation during mitosis due to the E3 ligase, Fbw7α. Fbw7α carries out its function after GSK3β- and CDK2/cyclin A2-dependent phosphorylation events on Thr171 and Ser175 of BLM which lies within a well-defined phosphodegron, a sequence which is conserved in all primates. Phosphorylation on BLM Thr171 and Ser175 depends on prior phosphorylation at Thr182 by Chk1/Chk2. Thr182 phosphorylation not only controls BLM ubiquitylation and degradation during mitosis but is also a determinant for its localization on the ultrafine bridges. Consequently lack of Thr182 phosphorylation leads to multiple manifestations of chromosomal instability including increased levels of DNA damage, lagging chromatin, micronuclei formation, breaks and quadriradials. Hence Thr182 phosphorylation on BLM has two functions-it regulates BLM turnover during mitosis and also helps to maintain the chromosomal stability.

MRN complex-dependent recruitment of ubiquitylated BLM helicase to DSBs negatively regulates DNA repair pathways

“…This mechanism of BLM recruitment to DSBs is in contrast to the processes characterized for its localization to the stalled replication forks 5 , where BLM recruitment predominantly depends RNF8/RNF168-mediated BLM ubiquitylation. Further these BLM ubiquitylation events differ from MIB1 and Fbw7α mediated polyubiquitylation of BLM, both of which lead to BLM degradation by 26S proteasome 32 , 33 .…”

“…Recently multiple mechanisms involving post-translation modifications of BLM have been deciphered. These include mechanisms which regulate the constitutive expression of BLM 40 , its interaction with TopBP1 and thereby conferring stability 33 , its turnover in both mitosis and G1-phase 32 , 33 and recruitment to PML-NBs 41 . It is quite possible that some these processes that regulate BLM cellular functions may act in close coordination with mechanisms by which BLM responds and ultimately repairs DSBs in different phases of the cell cycle.…”

“…All cells used were tested free from mycoplasma contamination. Synchronization of U2OS–AsiSi–ER cells in S- and G1-phase by double thymidine block was carried out as published 32 . Supplementary Figure 1 summarizes the growth conditions of U2OS–AsiSI–ER cells used to study the role of BLM in either recruitment phase or repair phase.…”

FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation

“…FBW7 could have multiple substrates involved in various DNA repair processes. For instance, it was recently shown that bloom helicase (BLM) is targeted by FBW7 during mitosis [59]. As FBW7 deficiency exhibits more profound cellular hypersensitivity to MMC compared with cells with the FAAP20 SA mutant, we do not exclude the possibility that FBW7 may have other substrates required for DNA ICL repair.…”

Mitochondrial functions of RECQL4 are required for the prevention of aerobic glycolysis-dependent cell invasion

“…Immunofluorescence, reverse-transcription PCR, matrigelinvasion assays, transmission electron microscopy Immunofluorescence for SOD2 and AcLys68 SOD2 was carried out as described (Kharat et al, 2015). Imaging was carried out by using a 63×/1.2 oil immersion objective.…”