We report here that Recql5-deficient mouse ES cells were hypersensitive to CPT in a replication-dependent manner. Yet, they were not more sensitive than their wild-type counterpart toward a number of other DNA-damaging agents that can also impede DNA replication. Moreover, this phenotype is also observed in Recql5-deficient MEF cells. Together, these data demonstrate the unique requirement of Recql5 for the optimal cell survival in both mouse ES and MEF cells after CPT treatment. In addition, we showed that after CPT treatment, a significant fraction of Recql5-deficient cells, but not their wild-type counterpart, lost their ability to incorporate BrdU in S phase. We noticed that this loss of replication competence during the S phase is followed by a rapid accumulation of DSBs and GCRs and then cell death. It has been demonstrated that CPT can cause the collapse of replication forks (Kohn and Pommier, 2000
; Strumberg et al., 2000
). DSB generated from such fork collapse is believed to represent the main cytotoxic lesion induced by CPT and its derivatives (Pommier et al., 2006
). Therefore, our data strongly suggest that Recql5 is required for maintaining the replication competency when forks are impeded after CPT treatment.
We proposed that in CPT-treated wild-type cells, a Recql5-dependent mechanism functions to maintain the integrity and the activity of DNA replication to prevent the collapse of the impeded replication forks. The dilemma caused by the replication impediment can then be resolved either by HR or by other mechanism(s), preventing the accumulation of DSBs. In contrast, in Recql5-deficient cells, the impeded replication forks are more susceptible to collapse, resulted in a significant accumulation of DSBs. This extension is consistent with our previous observation that Recql5-deficient cells exhibited both an elevated HR-mediated repair and an increased in DSB accumulation after CPT treatment (Hu et al., 2005
). Therefore, Recql5 has a nonredundant role with HR with respect to the optimal cell survival after CPT treatment. Thus, defects in either HR or the Recql5-related pathway can significantly increase the cells' sensitivity toward CPT and its derivatives. Importantly, the combined functions of these two mechanisms as well as other (Pommier et al., 2006
) are required to provide an optimal protection against CPT cytotoxicity (C).
It has been reported recently that under reconstituted conditions, camptothecin-induced Topo I–DNA complex formation resulted in the retardation of DNA uncoiling (Koster et al., 2007
). Furthermore, in yeast, this retardation of DNA uncoiling is associated with the impediment of both transcription and replication in vivo (Koster et al., 2007
). The implication that Recql5 can prevent impeded replication forks from collapsing after CPT treatment is in agreement with this recent finding and suggests that a similar scenario also exist in mammalian cells. A role of Recql5 in maintaining the integrity of stalled replication forks to prevent fork collapse is in agreement with previous reports that link this helicase (Garcia et al., 2004
; Kanagaraj et al., 2006
) and its homologues (Ozsoy et al., 2003
) to DNA replication. Interestingly, we found that Recql5-deficient cells that were initially incapable of incorporating BrdU could eventually resume DNA synthesis and reenter the next phase of the cell cycle after CPT is removed. This temporally cessation of DNA synthesis could reflect either a delay in the reactivation of the impeded forks or the inability of impeded forks to resume DNA synthesis, and therefore cells must rely on the firing of new origins of replication in order to complete the duplication of the genome. Future experiments will be needed to distinguish these possibilities. Intriguingly, this resumption of DNA replication in CPT-treated Recql5-deficient cells occurred within 4 h after CPT removal, when the proper repair or restoration of the collapse forks had not yet been completed, which would be consistent with a current model that the stalled replication fork, but not the specific cues that cause the stalling, provides the necessary signal for intra-S cell cycle arrest in mammalian cells (Zachos et al., 2005
). In any cases, this temporally cessation of DNA synthesis is associated with an increase in genome instability and a dramatic decrease in cell survival.
Human cells that are deficient in BLM or WRN and mouse cells deficient in Wrn are more sensitive to CPT and other DNA-damaging agents than their wild-type counterparts (Hook et al., 1984
; Ogburn et al., 1997
; Lebel and Leder, 1998
; Poot et al., 1999
; Rao et al., 2005
). Intriguingly, to our knowledge, only Recql5-deficient cells are uniquely hypersensitive to CPT. The molecular basis underlying this unique role of Recql5 remains unclear. It should be noted that that the structures of the stalled forks elicited by CPT and other Topo I inhibitors are likely to be distinct from those induced by other DNA-damaging agents or by nucleotide deprivation, the two other major causes of replication stalling. For instance, when a replication fork is stalled by a DNA damage lesion or due to nucleotide deprivation, the progression of the DNA polymerase is impeded, but the MCM replication helicase complex associated with the fork can continue to advance, leading to the creation of an extended stretch of single-strand DNA (ssDNA) region and the activation of intra-S checkpoint (Liu et al., 2000
; Byun et al., 2005
). In contrast, CPT causes the stalling of replication fork by stabilizing DNA-Topo I covalent complexes in front of advancing replication forks. Thus, it impedes the advancement of both the DNA polymerase and MCM helicase complex and hence does not directly create excessive amount of ssDNA. Moreover, DNA-Topo I covalent complexes similar to those induced by CPT also exist under normal physiological conditions. Thus, the formation of a DNA-Topo I covalent complex per se may not be consequential. Indeed, CPT causes a peculiar late S–early G2 cell cycle arrest rather than the typical intra-S arrest triggered by many other types of replication-inhibiting agents or treatments (Zhou et al., 2002
). Therefore, alternative molecular mechanism(s) must exist in order to maintain the integrity of the stalled forks induced by CPT. We hypothesize that Recql5 plays a critical role in such a mechanism. More importantly, when a replication fork is stalled by a DNA damage lesion, both the stabilization of the stalled fork and the subsequent successful repair of the damage lesion are likely to be necessary in order to preserve the integrity of the genome and to promote cell survival (Hook et al., 2007
). In contrast, when a replication fork is stalled as the result of Topo I poisoning, the removal of the inhibition may be the only key event for reactivating the stalled fork. Thus, we propose that in the event of Topo I poisoning by CPT, stabilization of the stalled replication forks by Recql5 alone provides the critical time that is required for the dissociation of CPT from the stalled fork and the restoration of the fork before it collapses (C, route 3-4-1R). Therefore, Recql5 is both necessary and sufficient for promoting cell survival after the exposure to CPT, but no other types of replication fork–stalling agents or treatments. However, CPT-induced stalled fork could also collapse, for instance, when it is not protected by Recql5 in Recql5
knockout cells or when the number of the stalled forks outpaced the capacity of Recql5. In this case, faithful HR repair is necessary for preserving the integrity of the genome and promoting survival. Failure to execute such a faithful repair can lead to genome instability or even cell death (C, route 3a-4a).
CPT and its derivatives represent a group of very promising anticancer agents, two of which, Topotecan and Irinotecan, have recently been approved by the FDA for treating patients with several types of malignancies (Pizzolato and Saltz, 2003
; Pommier et al., 2006
). However, despite a broad range of indication in treating many types of cancers, these drugs, when used in single-agent therapy, cannot provide a satisfactory curative effect. Therefore, it has become increasingly evident that combinatorial therapies involving multiple agents and/or treatment modalities is necessary in order to fully realize the potential of these drugs in anticancer treatments (Pommier et al., 2006
). A thorough understanding of the molecular determinants of drug sensitivity as well as the molecular pathways that are specific to camptothecins and other Topo I poisoning drugs will be critical in formulating such combinatorial treatment regimens. The mouse Recql5 and human RECQL5 are highly conserved (Ohhata et al., 2001
). Thus, it is expected that mutations in RECQL5
in human cells should also resulted in an hypersensitivity to CPT. In this context, the finding that Recql5 is an important determinant of CPT resistance can have important implication in improving the use of CPT derivatives and other Topo I inhibitors in anticancer treatment. In particular, we have shown that deletion of Recql5
in mice resulted in a significant increase in cancer susceptibility (Hu et al., 2007
). Thus, it is possible that RECQL5
mutations may also be associated with some human cancers. In that case, RECQL5 may be a valuable biomarker for determining whether Topo I inhibitors should be used in a patients' treatment regimen. Furthermore, we have noticed that the combination of Recql5 inactivation and CPT exposure is similar to the effect of PARP inhibitors, that is, both render mammalian cells more dependent on HR for survival in response to agents that can cause replication fork collapse. HR deficient cancer cells, for example, those with BRCA1 or BRCA2 deficiency, are hypersensitive to PARP inhibitors, and PARP inhibitors have been proven very effective in treating tumors that are deficient in either BRCA1 or BRCA2 (Bryant and Helleday, 2004
; Bryant et al., 2005
; Farmer et al., 2005
). Thus, RECQL5 may be a potential target for developing new anticancer drugs that can enhance the efficacy of Topo I inhibitors and/or those that target HR.