The genomic instability prevalent in human RecQ helicase disorders is characterized at the cellular level by gross chromosomal rearrangements. Studies of prokaryotes and eukaryotes indicate the importance of RecQ helicases to function in the DNA damage response by maintaining legitimate HR during replication of damaged DNA. In this study, we have investigated the importance of RECQL, one of the less-well-characterized RecQ helicases, for the preservation of genomic stability in mammalian cells. Embryonic fibroblasts from Recql knockout mice display chromosomal structural aberrations and aneuploidy, demonstrating for the first time that RECQL plays an important role in genome stability maintenance.
RECQL deficiency resulted in an increased sensitivity of the MEFs to IR which primarily induces DSBs. The elevated SCE observed in RECQL-deficient mouse cells (this study) and human cells (our unpublished results and reference 25
) may be due to unsuccessful attempts to “repair” damaged replication forks by HR at DSBs. The persistence of abundant spontaneously forming Rad51 foci and γH2AX, an early marker of DSB formation, in RECQL-deficient cells adds further support to a proposed role for RECQL in HR repair at sites of chromosomal DNA damage. Constitutively high numbers of Rad51 foci have also been detected in Werner syndrome and Bloom syndrome cells (38
). The presence of Rad51 foci corresponding to nucleoprotein filaments that are necessary for early strand invasion during HR suggests incomplete resolution of recombination intermediates as a consequence of the absence of RECQL.
The gross chromosomal rearrangements, translocations, and aneuploidy in cells of Recql−/− but not wild-type littermates indicates that the helicase is necessary for the proper maintenance of genomic stability. In spite of this, RECQL is not required for cell viability or proliferation and Recql knockout mice are viable, fertile, and apparently not tumor prone under normal colony conditions. However, it should be emphasized that small numbers of mice were evaluated in each group and no genetic or environmental carcinogenic stimuli were used in these initial studies.
The genetic background and/or tissue specificity of RECQL function may protect the mouse from severe abnormalities that might be expected from the magnitude of chromosomal aberrations detected at the cellular level. It will be critically important to evaluate RECQL deficiency in other genetic backgrounds, including mutations in genes encoding other RecQ helicases or DNA replication and repair proteins. In addition, challenging Recql knockout mice with DNA-damaging agents may expose phenotypes that will be helpful to elucidate a disease or a predisposition to cancer associated with RECQL deficiency.
One potential reason for the apparent lack of a phenotype in RECQL-deficient mice is functional redundancy, in which one or more of the other RecQ DNA helicases is able to compensate for the absence of RECQL. Expression levels of the known family members, WRN, BLM, RECQL4, and RECQL5, were not altered in Recql
knockout mice, suggesting that if one or more of these helicases compensates for the absence of RECQL, the normal level of expression is sufficient for this compensation. Recent studies carried out with the chicken B-lymphocyte DT40 cell line support the notion that there may be redundancy of function among the various RecQ helicases (52
The mixed genetic background of the mice may also have contributed to the apparent lack of a phenotype, illustrated by the finding that a WRN helicase domain knockout mouse induced accelerated tumorigenesis only in a p53
null background (23
). Finally, it remains possible that RECQL function is not critical under normal physiological conditions in the mouse but could be important in the response to external stressors such as environmental factors or genotoxins. The nearly 85% sequence homology of the mouse and human RECQL proteins suggests the two proteins are likely to have conserved functions; however, intrinsic differences between mice and humans can contribute to differences in tumor susceptibility. For example, WRN-deficient mice lack a disease phenotype (24
); however, late-generation telomerase- and WRN-deficient mice (mTerc−/− Wrn−/−
) displayed clinical symptoms of premature aging and the types of tumors typically observed in Werner syndrome patients (4
). It is plausible that the manifestation of disease phenotypes of RECQL deficiency in mice is related to telomere maintenance or some other event critical for genomic stability.
Having demonstrated that RECQL is important for chromosomal stability, we now face the challenge of understanding the potentially unique molecular functions of RECQL helicase to prevent illegitimate recombination during replicational stress. It is highly likely that RECQL cooperates with other nuclear DNA metabolic factors in this capacity. Strong candidates for protein interactors with RECQL that serve to suppress crossover of sister chromatids are recombination proteins that are involved in DNA replication or mismatch repair factors already known to regulate genetic recombination. In the absence of a functional RECQL helicase, DNA replication forks may not be maintained properly, giving rise to elevated levels of SCE (33
In addition to recombination proteins, type IA topoisomerases have been implicated genetically and biochemically in collaboration with RecQ helicases to preserve chromosomal integrity. BLM and Sgs1 helicases are proposed to act with type IA topoisomerases to regulate recombination levels in vivo by suppressing the formation of crossover products that accumulate from resolution of Holliday junction (HJ) intermediates (17
). A model for BLM to suppress crossovers was proposed on the basis of in vitro studies that demonstrated the unique ability of BLM with Topo IIIα to catalyze double HJ dissolution (57
). Although RECQL was reported to be associated with Topo IIIα in human cells (18
), the purified recombinant protein failed to substitute for BLM in the in vitro double-HJ dissolution reaction (55
). It is conceivable that in the cellular setting, RECQL acts upon a key DNA intermediate of HR with additional protein factors. The posttranslational state or protein interactions of RECQL with mismatch repair factors (e.g., MSH2/6, EXO-1) (9
) may be important for the role of RECQL in processing structures to prevent illegitimate recombination. The constitutively high number of Rad51 nuclear foci in RECQL-deficient cells further supports a role for RECQL in the disruption of inappropriately paired DNA recombination intermediates.
Although a genetic disorder has not been linked to a mutation in Recql
, recent analyses of Recql
single-nucleotide polymorphisms (SNPs) have identified an association of RECQL with a reduced survival of pancreatic cancer patients (26
). Only one RECQL A159C SNP allele is required to significantly decrease overall pancreatic cancer survival, suggesting a predictive and prognostic role for RECQL SNPs. RECQL SNPs displayed significant genetic interaction with SNPs in the HR repair genes ATM
, and XRCC3
. A role for RECQL in HR is further suggested by the observation that SNPs in RECQL result in a very poor response to the anticancer drug gemcitabine-induced radiosensitization that selectively requires functional HR (26
). Our results suggest that the chromosomal instability arising from RECQL deficiency may contribute to a predisposition to cancer. Microsatellite instability in the polyguanine repeat (G)9
in the Recql
gene is frequently observed in mismatch repair-deficient human nonpolyposis colorectal cancer (37
Loss of heterozygosity of 12p12, the chromosomal location of the Recq1
gene, is a frequent event in a wide range of hematological malignancies and solid tumors, suggesting the presence of a tumor suppressor locus. Allelic losses on chromosome 12p12-13 are associated with childhood acute lymphoblastic leukemia and several solid neoplasms (31
). Chromosome 12p12 deletion has been reported in a rare chronic myeloid leukemia-like syndrome case in a Li-Fraumeni syndrome family (14
). RECQL is highly expressed in the lungs (21
), and deletions at chromosome 12p12 have been reported in bronchial epithelia of patients with primary non-small-cell lung cancer (13
), suggesting that RECQL sequence alterations may influence the risk of lung cancer.
RECQL has not been directly linked to a human disease or cancer; however, the present study demonstrates that RECQL deficiency leads to spontaneous chromosomal instability and aneuploidy, both characteristics of cancer cells, suggesting a role for RECQL in a predisposition to cancer. Results obtained with RECQL-deficient mice provide a foundation from which to investigate the interactions of RECQL with other genetic factors or tumor suppressors that exist in vivo to preserve chromosomal integrity and prevent carcinogenesis.