Molecular mechanism of how ultrafine anaphase bridges contribute to chromosomal instability

Professor Chan, Ying Wai   (Principal Investigator (PI))

36

2021-01-01

986520

Molecular mechanism of how ultrafine anaphase bridges contribute to chromosomal instability

chromosomal instability, chromosome segregation, DNA repair, ultrafine anaphase bridge

Cell Biology

Biology and Medicine (M)

27110120

Early Career Scheme (ECS)

2020

Completed

1 Unresolved DNA entanglements formed between sister chromatids can persist in anaphase and give rise to a special type of DNA bridge, termed ""ultrafine anaphase bridges (UFBs)"". Although it has been proposed that UFBs serve as a driver of chromosomal instability (CIN), precise molecular mechanisms remain unclear. Our previous work showed that UFBs are processed in late anaphase/telophase to generate single-stranded DNA (ssDNA) bridges, which will eventually be broken at cell division to induce DNA damage and gross chromosome abnormalities in the following cell cycle. We hypothesise that the repair of DNA damage induced by UFB breakage is highly mutagenic due to the single-stranded nature of the bridge remnants. Therefore, we will determine the repair mechanisms of the UFB remnants. We propose that the DNA ends of the broken UFBs contain an extensive amount of ssDNA that possibly leads to G1 recombination. In addition, the ssDNA may be cleaved by nucleases, followed by ligation mediated by non-homologous end joining (NHEJ), leading to deletions and translocations. Our work will provide the first study of the detailed molecular mechanisms of how breakage of UFBs leads to CIN. 2 Failure to resolve UFBs by interfering with the functions of UFB-binding proteins, such as PICH (PLK1-interacting checkpoint helicase) or BLM (Bloom’s syndrome helicase) induces a significant increase in the frequency of chromatin bridges. Chromatin bridges often lead to micronuclei formation and occasionally induce a complete failure of cytokinesis. Therefore, proper resolution of UFBs is important for cells to maintain genome stability and proliferation. However, the origin of those chromatin bridges is currently unclear. We hypothesise that translocase activity of PICH cooperates with its binding partner, BLM helicase, to prevent the formation of chromatin bridges. To test our hypothesis, we will target PICH using our recently developed PICH degron cell line model, and investigate how impairing UFB resolution induces the formation of chromatin bridges and other cellular defects. 3 UFBs can originate from catenated dsDNA, late replication intermediates and recombination intermediates. Despite different underlying DNA structures, we previously demonstrated a common mechanism for their resolution: PICH recruits BLM helicase to unwind duplex DNA present in the UFBs to generate ssDNA bridges. We hypothesise that specific proteins are involved in processing different types of UFBs before the action of BLM or other enzymes. For instance, BLM and TOP3A can be recruited independently to PICH-coated UFBs, suggesting that they may play distinct roles in resolving different types of UFBs. Therefore, we will determine the precise mechanism of UFB resolution by focusing on the study of the interaction between PICH and BLM/TOP3A. We will also identify novel UFB-processing proteins by immunoprecipitating known UFB-binding proteins and identifying their interacting partners by mass spectrometry.