Given the genetic linkage of other human RecQ helicases (WRN, BLM, RECQ4) to diseases characterized by premature aging, cancer, and chromosomal instability, we investigated the significance of human RECQ1 for genome integrity, and more specifically its role in the DNA damage response. In this work, we have demonstrated that endogenous human RECQ1 becomes phosphorylated and re-localizes its sub-nuclear distribution to the chromatin fraction upon cellular exposure to DNA damage. Depletion of RECQ1 renders cells sensitive to IR or the topoisomerase inhibitor CPT, and results in spontaneous γ-H2AX foci and elevated SCE, indicating an accumulation of double strand breaks. The biological results suggest that RECQ1 either serves to prevent double strand breaks from forming or directly helps to repair double strand breaks through its interaction with HR repair proteins such as Rad51. Collectively, these studies provide the first evidence for a role of human RECQ1 in the response to DNA damage and chromosomal stability maintenance and point to the vital importance of RECQ1 in genome homeostasis.
The significant reduction in cell proliferation due to RECQ1 depletion in human cells is different from that observed in mice in which RECQ1 deficiency had no obvious effect on the growth/proliferation of mouse embryonic fibroblasts nor the normal development or postnatal growth of mice 
. The phenotypes of complete loss of human RECQ1 are likely to be more severe than those observed, particularly in the case of shRNA selection, since that protocol presumably selects for clones that fail to completely silence RECQ1. This allows the possibility that RECQ1 is essential for cell viability in humans, in contrast to the case in mice.
It is conceivable that manifestation of cellular or organismal phenotypes in Recql
-null mice may be masked by other genetic factors. For example, WRN
-null mice do not exhibit any phenotypes prevalent in WS; however, premature aging phenotypes are observed in a WRN
-null telomerase-knockout mouse characterized by the presence of critically short telomeres (for review, see 
). Furthermore, late generation mTerc-/- WRN -/-
mouse embryonic fibroblasts have an increased load of DNA damage and replicative senescence, properties similar to that observed in human WS fibroblasts.
In addition to their growth defects, RECQ1-depleted human cells display defective maintenance of the G2/M checkpoint following IR exposure and an aberrant apoptotic response to H2O2-induced stress. The role of RECQ1 in the apoptotic response may be either direct or the effect could be secondary, and the reduction in genotoxic stress-induced apoptosis could be due to the altered cell cycle distribution or viability of the RECQ1-depleted cells.
Intra-nuclear trafficking of nuclear proteins is an important aspect of the DNA damage response since DNA repair factors are known to localize to DNA damage foci. RECQ1 is predominantly found in the nucleoli of proliferating human cells and re-localizes to the nucleoplasm in response to IR-induced DNA damage or replicational stress induced by hydroxyurea. Like RECQ1, WRN and BLM also re-localize to the nucleoplasm after DNA damage 
; however, RECQ4 is constitutively present in nuclear foci irrespective of DNA damage 
, suggesting some differences in the sub-cellular distribution of human RecQ helicases before and after DNA damage.
Most of the RECQ1 is loosely tethered to the nucleus; however, a subset of RECQ1 molecules becomes resistant to detergent extraction after IR exposure, probably due to their tighter association with sites of damage in chromatin. Endogenous RECQ1 is phosphorylated in response to DNA damage or replicational stress, and preferentially associates with chromatin. A number of predicted phosphorylation sites for various stress-activated kinases exist in RECQ1. Our unpublished results indicate that RECQ1 is phosphorylated after DNA damage in AT mutated cells, suggesting that ATM is not the major candidate kinase. In vitro phosphorylation of RECQ1 by Protein Kinase C or Casein Kinase II modulates its catalytic activities (data not shown). RECQ1 and its protein partners may be localized to sites of DNA damage where RECQ1 utilizes its catalytic activities to process genomic DNA structures as a component of the DNA damage response.
In agreement with this, RECQ1 deficiency in both mouse 
and human cells resulted in an increased sensitivity to IR or CPT which introduce strand breaks that can be converted to double strand breaks during replication. Spontaneously elevated γ-H2AX foci and SCEs observed in RECQ1-depleted cells (this study and reference 
) may be due to unsuccessful attempts to ‘repair’ damaged replication forks by HR at double strand breaks. Thus, RECQ1 may have a direct role in repairing double strand breaks. Alternatively, RECQ1 may act to prevent double strand breaks from occurring in the first place. The latter explanation would be consistent with our observation that only a minority of RECQ1 co-localizes with γ−H2AX foci. The sensitivity of RECQ1 deficient cells to other DNA damaging agents remains unexplored and RECQ1 might also participate in other DNA repair pathways.
Strong candidates for RECQ1 protein interactors that serve to suppress cross-over of sister chromatids are recombination proteins, mismatch repair factors that regulate genetic recombination 
, and Type IA topoisomerases that have been implicated genetically and biochemically to collaborate with RecQ helicases to preserve chromosomal integrity 
. It is conceivable that Rad51 may act to regulate RECQ1 helicase activity on HR intermediates (D-loop, Holliday Junction) that RECQ1 has the ability to unwind 
since Rad51 plays a critical role in the strand invasion step of HR repair of strand breaks. However, we did not detect an effect of Rad51, inhibitory or stimulatory, on RECQ1 catalyzed unwinding of a variety of oligonucleotide-based DNA substrates including forked duplex, D-loop, or synthetic Holliday Junction (data not shown). RECQ1 may utilize its motor ATPase function to displace Rad51 from inappropriate strand invasion intermediates that are either homeologous or occur in an untimely manner. A similar function was proposed for the yeast Srs2 helicase which physically binds to Rad51 
and displaces the strand exchange protein from DNA 
Our observation that human RECQ1 directly interacts with Rad51 is consistent with other reports that RecQ helicases (BLM, Sgs1 
) function in DNA repair through HR by their physical interactions with Rad51. It was proposed that the physical interaction between BLM and Rad51 serves to recruit BLM to sites of recombinational repair and possibly load BLM onto Holliday Junctions in a particular orientation that would then dictate the direction of junction translocation. The spontaneously elevated nuclear Rad51 foci observed in BS cells 
may represent accumulation of unresolved recombination intermediates or a greater load of endogenous DNA damage in the absence of the RecQ helicase. Our earlier demonstration that RECQ1 interacts physically and functionally with mismatch repair proteins 
is likely to be important in the mechanisms whereby RECQ1 regulates genetic recombination. MSH2/6 proteins may interact with RECQ1 to unwind recombination intermediates that contain mismatches, as proposed for the mechanism of Sgs1 to prevent homeologous recombination 
It was suggested that the fission yeast Rqh1 helicase functions after Rad51 focus formation during DNA repair through its interaction with Topoisomerase III in G2 
; however, Rqh1 and other RecQ helicases may have early and late roles in homologous recombination 
. Human RECQ1 was reported to physically interact with Topo IIIα 
, and Rad51 focus formation after DNA damage is not dependent on RECQ1 in mouse cells 
. It is likely that RECQ1 utilizes its catalytic helicase and strand annealing activities to facilitate the resolution of HR repair intermediates with other proteins intimately involved in this process. The observed elevated SCE after CPT-induced DNA damage is consistent with this proposal.
BLM has been proposed to suppress SCEs by its concerted action with Top3α 
; however, RECQ1, WRN, and RECQ5β were not able to substitute for BLM in Top3α mediated double Holliday Junction dissolution on model DNA substrates in vitro 
. RECQ1−/− BLM−/−
or RECQ5−/− BLM−/−
DT40 cells displayed a growth defect, and RECQ5−/− BLM−/−
cells had a higher SCE frequency compared with BLM−/−
. An independent role of RECQ5 in sister chromatid exchange suppression was demonstrated in embryonic stem cells or differentiated fibroblasts from RECQ5
-knockout mice 
. Primary embryonic fibroblasts from the RECQ1 knockout mouse display spontaneously elevated SCEs and chromosomal aberrations 
, suggesting that RecQ helicases participate in non-redundant pathways to suppress cross-overs during mitosis.
Although a genetic disorder has not yet been linked to a mutation in RECQ1, recent analyses of RECQ1
single nucleotide polymorphisms (SNPs) have identified an association of RECQ1 with a reduced survival of pancreatic cancer patients 
. RECQ1 SNPs displayed significant genetic interaction with SNPs in the homologous recombinational repair genes ATM, RAD54L, XRCC2 and XRCC3. A role of RECQ1 in HR is further suggested by the observation that SNPs in RECQ1 affect the response to the anti-cancer drug gemcitabine induced radio-sensitization that selectively requires functional HR 
. The chromosomal instability arising from RECQ1 deficiency may contribute to a cancer predisposition. RECQ1 is differentially up-regulated in transformed cells or cells that are actively proliferating 
. We propose that RECQ1 confers genomic integrity in transformed or actively proliferating cells.
The human RECQ1
gene is localized to chromosome 12p11-12 
, a location of instability in testicular germ-cell tumors 
expression in mouse is highest in the testis 
. The purification of a RECQ1 ribonucleoprotein complex from rat testis with testis-specific small non-coding piRNAs involved in gene silencing, cell growth, and development 
raises the question of what is the precise mechanism whereby RECQ1 and its associated piRNA complex regulates the germ-line. The mechanistic similarities between piRNA synthesis and DNA replication 
suggest that RECQ1 and its associated DNA replication/repair proteins (RPA, EXO-1, MSH2/6) are involved in piRNA biogenesis. This is an unexpected and potentially exciting role for RECQ1 protein in the metabolism of regulatory small RNAs important for development and mechanisms of inheritance. Presently, it can only be speculated if the piRNA complex regulates the genome at the level of DNA or histones, or at a posttranscriptional level. Interestingly, QDE-3, the RECQ1 homolog in Neurospora crassa
, has also been implicated in gene silencing 
. The qde-3
mutant was found to be hypersensitive to a variety of DNA mutagens and exhibit increased mutability and extensive chromosomal deletions 
, implicating a broader role of the Neurospora
RECQ1 homolog in the DNA damage response and chromosomal stability. Our cellular studies implicate a unique and important role of human RECQ1 in genome homeostasis as well. Further work is required to understand RECQ1-piRNA genome defense association. Clearly, RECQ1 has become the latest member of the RecQ helicase family with important biological functions through its involvement in the DNA damage response and fundamental processes of chromosomal nucleic acid metabolism.