Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation

Abstract

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

Conserved function ofDrosophilaFancd2 monoubiquitination in response to double-strand DNA breaks

“…These studies have linked Fancd2 to organismal viability, germ cell function, and cancer predisposition in the absence of DNA damage. Further, mirroring findings in human cells, Fancd2 mutants in mice, zebrafish, Caenorhabditis elegans , and Drosophila , all compromise organismal viability in response to DNA damaging ICL or DSB agents ( Houghtaling et al 2003 ; Marek and Bale 2006 ; Lee et al 2007 , 2010 ; Vinciguerra et al 2010 ; Bretscher and Fox 2016 ; Ramanagoudr-Bhojappa et al 2018 ; Yang et al 2019 ; Germoglio et al 2020 ; Clay et al 2021 ). The conservation of critical FA proteins across various species emphasizes the importance of the pathway in maintaining genomic stability.…”

POLQ suppresses genome instability and alterations in DNA repeat tract lengths

“…The converse may be true for POLQ – / – clones undergoing a telomere-driven crisis, so that cell cycle progression and population doubling rates may be expedited, with reduced opportunity for corrective DNAR that safeguards genomic integrity. As POLQ plays an important role in the rescue of intermediates of aborted HR that materialize late in the cell cycle ( 22 ), frustration of TMEJ may also deter cell cycle pausing pre-mitosis, so that chromosomal damage persists ( 98 , 99 ), is extruded into micronuclei (MN) ( 100 ) or triggers apoptosis without suspension of proliferation. In concert with the skewed distributions of inter- and intra-chromosomal telomere fusions (Figure 3A ), POLQ deficiency supports long-range DNA recombinations that precipitate genomic heterogeneity ( 100 ), manifest in the polyclonal post-crisis populations ( Supplementary Figure S5 ).…”

“…As POLQ plays an important role in the rescue of intermediates of aborted HR that materialize late in the cell cycle ( 22 ), frustration of TMEJ may also deter cell cycle pausing pre-mitosis, so that chromosomal damage persists ( 98 , 99 ), is extruded into micronuclei (MN) ( 100 ) or triggers apoptosis without suspension of proliferation. In concert with the skewed distributions of inter- and intra-chromosomal telomere fusions (Figure 3A ), POLQ deficiency supports long-range DNA recombinations that precipitate genomic heterogeneity ( 100 ), manifest in the polyclonal post-crisis populations ( Supplementary Figure S5 ). Chromosome segregation defects may result in ploidy increments in POLQ-deficient cells that contribute to subclonal diversification ( 101 ) through juxtaposition and amplification of extra-chromosomal DNA, including ALRs and co-regulated genes ( 102 , 103 ).…”

DNA Damage Responses during the Cell Cycle: Insights from Model Organisms and Beyond

“…In addition to the use of chemical mutagens and irradiation as experimental sources of DNA damage, site-specific DSB systems including CRISPR/Cas9 have been used to great effect to study Drosophila DDRs [ 36 , 37 ]. Additionally, our group has exploited the salt excretion function of Drosophila rectal papillar cells to screen for DDR genes [ 38 , 39 , 40 ], which relies on a mechanism to respond to DNA breaks in mitosis (discussed in the section “ The DDR during mitosis ”). By combining site-specific DSBs with feeding animals a high salt (NaCl) diet, mutants that specifically impact mitotic DSBs can be identified in Drosophila papillar tissue.…”

“…Papillar cells are polyploid (see “DDRs during and after endoreplication cycles”) and lack a requirement for p53 or CHEK family kinase signaling in response to DSBs. Instead, chromosome fragment segregation in this cell type relies on the alt-EJ regulator PolQ and members of the conserved Fanconi Anemia DNA repair family ( Figure 2 C) [ 39 , 40 ]. Our findings may mirror those of mammalian cells, where Fanconi Anemia proteins localize to regions of the genome that are sites of mitotic DNA synthesis (MIDAS) [ 118 ].…”