FANCJ is essential to maintain microsatellite structure genome-wide during replication stress

Abstract

Microsatellite DNAs that form non-B structures are implicated in replication fork stalling, DNA double strand breaks (DSBs) and human disease. Fanconi anemia (FA) is an inherited disorder in which mutations in at least nineteen genes are responsible for the phenotypes of genome instability and cancer predisposition. FA pathway proteins are active in the resolution of non-B DNA structures including interstrand crosslinks, G quadruplexes and DNA triplexes. In FANCJ helicase depleted cells, we show that hydroxyurea or aphidicolin treatment leads to loss of microsatellite polymerase chain reaction signals and to chromosome recombination at an ectopic hairpin forming CTG/CAG repeat in the HeLa genome. Moreover, diverse endogenous microsatellite signals were also lost upon replication stress after FANCJ depletion, and in FANCJ null patient cells. The phenotype of microsatellite signal instability is specific for FANCJ apart from the intact FA pathway, and is consistent with DSBs at microsatellites genome-wide in FANCJ depleted cells following replication stress.

Common Chemical Inductors of Replication Stress: Focus on Cell‐Based Studies

“…HU is used for cell cycle synchronization [269], replication fork stability studies [249,252], studies of recovery mechanisms after the release of RS [242] and checkpoint responses [241]. Lower concentrations are used for RS induction [254], induction of senescence [74], apoptosis [257], and repair pathways induction [217]. HU reaches plasma concentrations around 0.1 mM; this should be bear in mind when interpreting the data for clinical relevance [261].…”

Replication stalling and DNA microsatellite instability

“…Interestingly, replication stress in FANCJ null patient fibroblasts, as well as in FANCJ knockdown cells, indicated that DSBs occurred under replication stress at diverse endogenous microsatellite loci (repeats of CTG (DMPK, ATXN1, ERDA1, TCF4), ATTCT (ATXN10), Pu/Py (PKD1), G quadruplex consensus (TUBA1B, HBB, MYC), and poly-T (BRIP1/FANCJ, TP53, TP63)) (Fig. 4) [92]. Equivalent treatment of cells knocked down for other proteins in the Fanconi anemia pathway, or fibroblasts from patients carrying mutations in other Fanconi anemia proteins (FANCA, -C, -D1, -D2, -I, -M, -Q, -P) did not show this effect.…”

“…Thus, non-B DNA forming microsatellites co-localize with hotspots of double strand breaks (DSBs), indels, and rearrangements [83,87–91], chromosome translocation junctions map to non-B DNA sites [92], and rare fragile sites co-localize with CTG microsatellites at the SCA1, 6, 7, 8, 12, and 17 loci [93–96]. In yeast and human cells expanded CNG tracts partially slow replication forks [5,64], and engage the DNA damage checkpoint machinery [77,97,98].…”

“…In yeast and human cells expanded CNG tracts partially slow replication forks [5,64], and engage the DNA damage checkpoint machinery [77,97,98]. Replication-induced DSBs have also been attributed to AT-repeat sequences at the ATXN 10 locus[92,99], common fragile site 16D [89,100], and H-DNA from the BCL-2, Friedrich’s ataxia and PKD1 loci [82,96,101]. …”

“…When replication was slowed by prolonged treatment with low concentrations of hydroxyurea or aphidicolin, small pool PCR across the repeat loci indicated that double strand breaks had occurred within 85 bp of the repeats (Fig. 3) [92], and sequencing of ectopic site translocation junction points demonstrated that DSBs had occurred within the CNG tracts (J. Barthelemy, unpublished). Interestingly, replication stress in FANCJ null patient fibroblasts, as well as in FANCJ knockdown cells, indicated that DSBs occurred under replication stress at diverse endogenous microsatellite loci (repeats of CTG (DMPK, ATXN1, ERDA1, TCF4), ATTCT (ATXN10), Pu/Py (PKD1), G quadruplex consensus (TUBA1B, HBB, MYC), and poly-T (BRIP1/FANCJ, TP53, TP63)) (Fig.…”