Here we show that conditions of replication stress, specifically aphidicolin-induced DNA polymerase α, δ and inhibition, and hydroxyurea-mediated inhibition of ribonucleotide reductase, induce a preferential accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, we find that RECQ1 is enriched at two major fragile sites, FRA3B and FRA16D, where replication forks have stalled following aphidicolin treatment. Moreover, RECQ1-depletion results in diminished checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability. These results suggest that RECQ1 is important for maintaining genomic integrity when DNA replication forks are slowed by hydroxyurea or aphidicolin and promote efficient recovery from replication stress.
Results from Thangavel et al proposed that RECQ1 is assembled at origins at the start of bidirectional replication and is subsequently lost from the origin perhaps still tracking along with the newly formed replisome following origin firing [34
]. Consistent with this, we found a significant enrichment of RECQ1 at origins when replication fork progression was inhibited by treatment with hydroxyurea or aphidicolin. Furthermore, our ChIP data show differential recruitment of RECQ1 at the lamin B2 origin in response to treatment with aphidicolin (about 30-fold) or hydroxyurea (about 11-fold). Aphidicolin has been shown to have little effect on the activation or initiation of replication origins and induces uncoupling of replication machinery [57
]; it is likely that RECQ1 recognizes the long stretch of single-stranded DNA produced due to functional uncoupling of the replicative polymerase and helicase complexes following aphidicolin treatment. Indeed, we found that endogenous RECQ1 displays preferential binding to CFS as compared to non-fragile control DNA especially after cells are treated with aphidicolin. CFS represent single-stranded unreplicated chromosomal regions caused by stalled or collapsed replication forks [5
]. This notion has been substantiated by investigation of replication timing [58
] and the evidence of involvement of checkpoint proteins ATR [59
], BRCA1 [61
], SMC1 [62
] and FANCD2 [63
] in fragile site stability. Additionally, CFS sequences including FRA16D are characterized by high AT content, AT-rich mini satellite repeats and their tendency to form secondary structures [64
]. Human RecQ proteins have demonstrated ability to resolve a variety of non-B DNA secondary structures [65
]. It is yet unknown whether RECQ1, like WRN [16
], can resolve the predicted cruciform structures at FRA16D that stall replication fork progression and contribute to chromosome breakage; however, the fact that RECQ1 was also enriched at FRA3B that is devoid of mini- or microsatellite [6
] indicates that the secondary structures alone may not be a sufficient structural element for RECQ1 binding to the fragile sites. WRN functions at fragile sites are critical but it has not yet been shown whether WRN is recruited to the fragile site loci in vivo, and whether it can distinguish fragile and non-fragile regions under replication stress. RecQ proteins are known to form multi-protein complexes to execute their functions [14
], and it is conceivable that yet unknown protein partners of RECQ1 recruit and/or mediate its functions at stalled and broken forks at fragile site loci and elsewhere in the genome.
Stalled replication fork activates checkpoint signaling pathways to coordinate cell cycle progression with repair of damage, ensuring the integrity of the genome [52
]. ATR and Chk1-dependent checkpoints prevent excessive formation of DNA double strand breaks during replication arrest [54
]. RPA protein complex, consisting of three subunits RPA72, RPA32 and RPA14, is a first sensor of replication-associated damage and is thought to signal activation of ATR and thereby trigger an intra-S checkpoint [68
]. Depletion of RECQ1 led to spontaneous phosphorylation of RPA32 and activation of Chk1; however, RECQ1-depleted cells are defective in triggering replication stress response and exhibit sensitivity to replication blocking agents [35
]. During normal DNA replication, optimal binding of RECQ1 to the origins may ensure appropriate and accurate genome duplication during S-phase. Loss of RECQ1 leads to aberrant elongation of progressing replication forks [34
] which may lead to activate the checkpoint response. The fork promoting activity of RECQ1 could be especially important at naturally occurring DNA sequences such as fragile sites that are at increased risk for stalling the replication fork even in the absence of external replication stress. Our data implicate that RECQ1 also participates in relaying signals of fork stalling that help coordinate a faithful cell cycle and recovery from replication stress. RECQ1 may contribute to mutational avoidance in unperturbed and deliberately stalled replication.
Chromosome breaks observed upon acute depletion of RECQ1 is consistent with a recent finding that depletion of RECQ1 activates DNA damage signaling cascade and accumulates replication-induced double strand breaks [35
]. Conditions that slow replication along the entire genome, such as aphidicolin treatment, lead to double strand break formation as a result of fork stalling and collapse at fragile sites and activate the double strand break repair pathways [69
]. Homologous recombination is a major mechanism utilized to repair stalled or collapsed replication forks [70
]. Importantly, homologous recombination mechanisms triggered by replication arrest differ from those involved in repairing classical two ended DNA double strand breaks [71
]. Thus, although the repair of I-Sce induced double strand break was not significantly modulated by RECQ1-deficiency [47
], a role for RECQ1 in recombination repair of replication-induced double strand breaks remains possible. In vitro activity of RECQ1 to unwind synthetic replication fork and catalyze strand exchange indicates its potential ability to form and subsequently branch migrate Holliday junction like recombination structures generated during template switching at the stalled forks [35
]. It is conceivable that in the absence of RECQ1, recombinogenic DNA structures at arrested forks are repaired via homologous recombination.
Fragile loci often coincide with chromosomal breakpoints in tumors [5
]. Given the elevated proliferation status of tumor cells, proteins involved in the cellular response to replication stress are likely to act as caretakers of the genome during tumor development [12
]. A mutation in RECQ1 has not been linked to a human disease yet, but an Oncomine database search shows that RECQ1 is over-expressed in many clinical cancer samples compared to matched normal samples (Additional file 1
: Figure S1). Similar trend of RECQ1 over-expression across various tumors is presented by another web database search (http://medicalgenome.kribb.re.kr/GENT/
). It is plausible that cancer cells position RECQ1 on to specific genomic loci so as to cope with increased replication-mediated DNA damage during rapid cell division; in normal cells, RECQ1 can act as a tumor suppressor by facilitating DNA repair and preventing mutations. This notion is consistent with the observation that RECQ1 is uniquely important for the proliferation of cancer cells [37
]. DNA breakage within CFS is thought to be a consequence of failing to complete replication and/or resolving the arrested forks prior to the end of telophase and chromosome segregation [19
]. Recent reports have also suggested that chromosomal breaks occur at fragile sites because these loci are late replicating and origin poor [73
]. Whether chromosomal breaks in RECQ1-deficient cells occur at CFS remains to be examined, but recruitment of RECQ1 at FRA3B and FRA16D suggests that RECQ1 either prevents replication fork stalling within origin poor regions or resolves replication problems at these CFS. Future experiments will examine what functional sub-modules of DNA replication are associated with RECQ1 at specific genomic loci and how it participates in dynamic response to challenges during DNA replication. Present findings together with the in vitro results indicate that impaired response to replication stress contributes to genomic instability in RECQ1-deficient cells.