Probing Genome Maintenance Functions of human RECQ1

The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in protecting the genome stability in all kingdoms of life. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β. Although the individual RecQ-related diseases are characterized by a variety of clinical features encompassing growth defects (Bloom Syndrome and Rothmund Thomson Syndrome) to premature aging (Werner Syndrome), all these patients have a high risk of cancer predisposition. Here, we present an overview of recent progress towards elucidating functions of RECQ1 helicase, the most abundant but poorly characterized RecQ homolog in humans. Consistent with a conserved role in genome stability maintenance, deficiency of RECQ1 results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased DNA damage and greater sensitivity to certain genotoxic stress. Delineating what aspects of RECQ1 catalytic functions contribute to the observed cellular phenotypes, and how this is regulated is critical to establish its biological functions in DNA metabolism. Recent studies have identified functional specialization of RECQ1 in DNA repair; however, identification of fundamental similarities will be just as critical in developing a unifying theme for RecQ actions, allowing the functions revealed from studying one homolog to be extrapolated and generalized to other RecQ homologs.


RECQ Helicase
The RECQ (also known as RECQL or RECQL ) gene resides on chromosome 2p 2 and encodes a 649 amino acid protein with a molecular mass of 73 kDa . RECQ protein is smallest of the human RecQ homologs, and shares maximum homology to the prototype E. coli RecQ. RECQ is a DNA-stimulated ATPase and helicase [ 0-]. Phylogenetic analysis of RECQ with closely related proteins reveals structural divergence (Figure 2).
A further insight into the anticipated biological roles of RECQ emanates from reviewing its structure-function relationship and molecular interactions.

CSBJ
Abstract: The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in protecting the genome stability in all kingdoms of life. Human RecQ homologs include RECQ , BLM, WRN, RECQ4, and RECQ5β. Although the individual RecQ-related diseases are characterized by a variety of clinical features encompassing growth defects (Bloom Syndrome and Rothmund Thomson Syndrome) to premature aging (Werner Syndrome), all these patients have a high risk of cancer predisposition. Here, we present an overview of recent progress towards elucidating functions of RECQ helicase, the most abundant but poorly characterized RecQ homolog in humans. Consistent with a conserved role in genome stability maintenance, deficiency of RECQ results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased DNA damage and greater sensitivity to certain genotoxic stress. Delineating what aspects of RECQ catalytic functions contribute to the observed cellular phenotypes, and how this is regulated is critical to establish its biological functions in DNA metabolism. Recent studies have identified functional specialization of RECQ in DNA repair; however, identification of fundamental similarities will be just as critical in developing a unifying theme for RecQ actions, allowing the functions revealed from studying one homolog to be extrapolated and generalized to other RecQ homologs. Members of the RecQ family have many structural motifs that are conserved from bacteria through humans. Besides the core helicase domain, most members possess RecQ C-terminal (RQC) and Helicase and RNase D C-terminal (HRDC) domains that mediate interactions with nucleic acids and proteins. A few RecQ proteins have acidic regions that are responsible for protein-protein interactions. WRN and FFA-1 proteins are unique in that they also contain an exonuclease domain. Total number of amino acids in each protein is indicated on the right and the full color scheme is indicated. Sequence of each protein in FASTA format was used as input to an online server called MyHits (http://myhits.isb-sib.ch/) for the mapping of various motifs in each protein.   Figure 1. Two RecA-like domains are denoted as RecA1 and RecA2. A. The nucleotide-binding pocket. ADP forms extensive contacts with RecA1 and less with RecA2; residues in contact with ADP are shown as sticks in pink color. B. RecQ-specific Zinc-binding module in RECQ1. A single Zn 2+ ion is coordinated by four Cys residues positioned on two antiparallel αhelices. C. The winged-helix (WH) domain of RECQ1. β-hairpin is shown in the upper-left corner and the important aromatic residue Tyr 564 is highlighted in red color. The figure was generated using PyMOL (http://www.pymol.org/). RECQ unwinds DNA with a 3'-5' polarity [ 2] and needs a 3′single strand DNA tail to unwind the substrate [ ]. RECQ unwinds standard duplex DNA substrates such as forked duplex, 3'overhang or 3'-flap, 5'-flap, and synthetic replication fork structures; these substrates signify model replication and repair intermediates lacking single strand character in the 3', 5', or both arms adjacent to the DNA duplex [ , 3-6]. Apart from conventional helicase activity, RECQ , like BLM and WRN, also promotes branch migration of Holliday junction (HJ) and D-loops in an ATPdependent fashion [4][5][6]. RECQ unwinds three-stranded D-loop with either a distended single stranded 3'-or 5'-tail by releasing the invading third strand from D-loop structures although a D-loop with protruding single stranded 3'-tail is a preferred substrate for unwinding [ 4]. In contrast to BLM helicase, RECQ is unable to unwind a DNA-RNA hybrid, catalyze fork regression, or displace plasmid D-loops lacking a 3'-tail but can unwind four-armed synthetic HJ structures that lacked a homologous core [4][5][6]. Unlike other known branch migration proteins such as BLM helicase and RAD54 both of which show no significant preference in directionality of branch migration, RECQ specifically catalyzes unidirectional branch migration, which may be instrumental in specific disruption of toxic, nonproductive intermediates of homologous recombination (HR) during DNA double strand break (DSB) repair in vivo [ 7]. However, RECQ is unable to use its motor ATPase to strip RAD5 from DNA during HR repair [ 8]. RECQ is also incapable of displacing streptavidin from a biotinylated oligonucleotide [ 9]. Consistent with a 3'-5' directionality of RECQ translocation on DNA, RECQ helicase activity is inhibited in a strand-specific manner by an alkyl phosphotriester modification to the sugar-phosphate backbone in the predicted translocating strand [20]. Moreover, the inability of RECQ to unwind G-quadruplex substrates differentiates this protein from other RecQ helicases including WRN, BLM, Sgs , or E. coli RecQ [ 5,2 ]. Specific functions of RECQ in DNA metabolism are not yet clearly understood but the reported disparity in helicase substrate preference suggests functional specialization.
In addition to DNA unwinding, RECQ promotes annealing of complementary single strand DNA in an ATP-independent manner [ 4]. ATP binding induces a conformational change in RECQ switching it from a strand-annealing protein to a DNA unwinding activity [ 4]. Further studies have suggested that distinct biochemical activities of RECQ are dictated by different oligomeric states modulated by single strand DNA and ATP binding [22]. Overall, strand-annealing appears to be an intrinsic property of the human RecQ family since it is conserved in WRN, BLM, .
RecQ helicases share a centrally located helicase domain that couples nucleotide hydrolysis to DNA unwinding and defines the RecQ family ( Figure ). Other conserved domains include RQC (RecQ C-terminal) and HRDC ((helicase and RNaseD C-terminal), missing in RECQ ) domains, which are implicated in protein interactions and DNA binding [26][27][28]. The N-and C-terminal extensions in eukaryotic RecQ helicases are poorly conserved but are shown to mediate protein-protein interactions [29][30]. Crystal structure of human RECQ protein lacking the first 48 and the last 33 amino acid residues (RECQ 49-6 6 ) was recently reported by Gileadi's group (Figure 3) [3 ]. Previously it was shown that the higher oligomeric structures formed by means of N-terminus region are responsible for DNA annealing activity of RECQ [ 5,22]; consequently, truncated RECQ 49-6 6 failed to promote strandannealing despite exhibiting unwinding of a forked-duplex comparable to the full-length RECQ [3 ]. Moreover, the Nterminal region of RECQ , either direct or through the formation of higher order oligomers, also appears to be critical for the dissolution of HJs [ 5,3 ]. Importantly, WRN protein was also shown to bind HJ and replication fork structures as an oligomer although whether this requires the N-terminal of WRN is not reported [32].

RECQ Signature Helicase Domain:
Core helicase domain of RECQ (amino acid residues 63-4 8) contains the seven signature motifs of superfamily 2 (SF2) helicases and harbors the ATP-binding pocket surrounded by highly conserved residues [3,33]. Mutational analyses in RecQ homologs and other SF2 helicases have revealed the necessity of these amino acids for nucleotide binding as well as for functions related to nucleotide binding activity [34][35][36]. We and others have shown that ATP binding regulates the helicase and strand-annealing activities of RECQ [ 4,22]. The overall fold of X-ray structure of RECQ bound to ADP and Mg 2+ in the absence of nucleic acid was found to be comparable to other SF2 helicases having signature structure of two RecA-like domains containing the seven motifs, and retaining the general helicase fold (Figure 3) [3 ]. The relative orientation of the two RecA-like domains in multiple crystal forms of RECQ was found to be changed significantly suggesting a higher degree of flexibility between these two domains [3 ]. Previous studies have reported that E. coli RecQ shows slight relative rotation of the helicase domain in nucleotide-bound form compared to the unbound state and as a result of this rotation, motif I acquires an open conformation that allows the nucleotide to enter into its designated cavity [36]. Motif 0 in human RECQ , located at the N-terminus of motif I is a RecQ-specific variant of the conserved Q motif in DEAD-box helicases and is implicated in ATP binding and hydrolysis [37]. The crystal structure of nucleotide-bound RECQ showed that the adenine moiety of ADP is hydrogen bonded to Gln96 within the motif 0 ( Figure 3) [3 ]. Notably, amino acid substitution of Gln in Motif 0 of RecQ5β and BLM has been reported to reduce the ATPase and ATPase/helicase function, respectively [30,34].

RQC Domain of RECQ :
RQC domain in RECQ is composed of a Zn-binding module and a helix-turn-helix fold called winged-helix (WH) domain. The Zn-binding module of RQC domain is practically identical between bacterial and human enzymes; in RQC domain of human RECQ , a Zn 2+ ion is coordinated by four Cys residues positioned on two antiparallel α-helices (Figure 3)  Furthermore, a unique tyrosine residue (Tyr564) at the tip of a βhairpin structure within the WH domain of RECQ is suggested to control helicase activity to unwind a simple fork duplex independent of its DNA-dependent ATPase activity (Figure 3) [3 ]. Vindigni's group has reported that the β-hairpin in the WH domain of RECQ is essential for DNA unwinding and oligomer formation [4 ]. The mismatch recognition complex MSH2/MSH6 stimulates RECQ helicase activity whereas RECQ stimulates the incision activity of human . Thus an antirecombination function of RECQ may involve suppression of homeologous recombination in conjunction with mismatch repair factors that specifically bind base pair mismatches [24][25]. In recent years, EXO-has emerged to be critical in helping cells deal with stalled replication forks [70-72] and interaction of RECQ with EXO-may also be important in this capacity. Another key protein partner of RECQ appears to be PARP- [57,65]. We first identified a direct protein interaction of RECQ with PARP-and demonstrated that RECQ -PARP--RPA associate in a common protein complex [57]. A conceivable biological function of this complex might be to regulate opposing activities of RECQ that are known to be regulated by RPA and ATP [ 4]. Given the ability of PARP-to modulate cellular ATP pools [73], interaction with PARP-may be important in providing an appropriate microenvironment to regulate dual activities of RECQ as necessary for the given cellular context. Moreover, RECQ and PARP-are also capable of interacting with the common components of mismatch repair system [54,74] indicating a possible role in the suppression of homeologous recombination between diverged sequences. Molecular basis for the elevated sister chromatid exchanges (SCEs) in RECQ deficiency is not yet understood, but its interactions with mismatch repair proteins (MLH /PMS2, MSH2/MSH6, and EXO-) and PARP-may be relevant in this regard. Vindigni's lab has recently identified an exclusive role of RECQ -PARP-interaction in the process of replication restart following Topoisomerase (TOP ) inhibition [65]. They have demonstrated that RECQ preferentially catalyzes fork reversal and promotes resetting of replication forks in vitro. Following TOP inhibition by Camptothecin (CPT) treatment, the poly(ADP)ribosylation activity of PARP-inhibits fork reversal by RECQ in vivo [65]. Thus, RECQ -PARPcomplex stabilizes regressed forks until repair of the TOP cleavage complex is complete thus preventing premature restart of regressed forks [65]. PARP activity is not required in RECQ -depleted cells as they cannot promote fork restoration in the absence of RECQ [65]; these cells likely employ HR for restart of replication following CPT treatment. Other known members of RECQ complex with 65] along with certain nucleosomal histones [65]. Interestingly, RECQ was also found to be present with PARP-and Ku80 in a multi-protein complex of APLF (APTX-PNK-Like Factor) which is implicated in recruitment of non-homologous end joining (NHEJ) proteins at DSBs [75][76][77][78]. Subsequently, we have shown that RECQ exhibits a direct physical interaction with the Ku70/80 subunit of DNA-PK complex and modulates in vitro end joining of DSBs [63]. Collectively, these studies have provided a framework for understanding the biological roles of RECQ though it is still premature to propose a comprehensive model for RECQ in the context of its catalytic functions and protein interactions. Identification of additional constituents of the RECQ -containing protein complexes and elucidation of how they modulate its biochemical activities will be critical to establish precise roles of RECQ in pathways of replication restart and DNA strand break repair.
Reported cellular phenotypes of RECQ -deficiency in mouse and humans implicate unique requirement of RECQ in genome stability maintenance [24,44,79]. Despite the lack of a phenotypic defect in unstressed RECQ knockout mice, primary embryonic fibroblasts from RECQ knockout mice display chromosomal instability [79]; and increased chromosomal instability is also observed upon depletion of RECQ in human cells [44]. Cellular deficiency of RECQ is characterized by spontaneously elevated SCEs [24] which are reminiscent of recombinogenic structures proposed to arise during replication restart following fork collapse [80]. Indeed, RECQdeficient cells accumulate DNA damage and display increased sensitivity to DNA damaging agents that induce stalled and collapsed replication forks [44,65,8 -82]. Furthermore, RECQ -deficiency in mice [79] or human cells [44] results in heightened sensitivity to ionizing radiation, which also causes oxidative DNA damage.
Our recent findings provide preliminary evidence for a unique role of RECQ in repair of oxidative DNA damage [57]. When cells are exposed to hydrogen peroxide (H2O2), RECQ is among the first RecQ proteins to arrive on chromatin and remains associated for the period of time required for the repair of strand breaks [57]. Remarkably, the chromatin localization of RECQ is more robust and rapid than that of WRN helicase which has been shown to function in oxidative DNA damage repair [57]. It is plausible that Roles of RECQ1 helicase the activities of these RecQ proteins are assigned to dedicated pathways or sub-pathways of oxidative DNA damage repair [83][84]. RECQ and its protein partners may be part of the DNA damage response by localizing to sites of oxidative lesions where they execute catalytic functions, alone or in concert. Consistent with this notion, purified recombinant RECQ catalyzes unwinding of duplex DNA containing oxidative base lesion such as thymine glycol, and the presence of RPA stimulates DNA unwinding by RECQ when the thymine glycol is positioned in the nontranslocating strand for the helicase [85]. RECQ -depleted cells rely on PARP activity for the repair of H2O2-induced DNA damage and when RECQ is deficient, these lesions are possibly repaired by an alternative mechanism that involves increased activation of PARP [57]. In contrast, WRNdeficient cells fail to activate PARP in response to oxidative damage [57]. These observations suggest a novel and non-overlapping role of RECQ in exogenously induced-oxidative DNA damage repair via modulation of PARP-; but a direct role of RECQ in specific pathway of oxidative DNA damage repair remains to be elucidated.
PARP-is known to bind to DNA strand breaks and subsequently synthesizes and transfers poly(ADP-ribose) polymers to itself and various nuclear proteins [86]. BLM or WRN-depleted cells exhibit constitutively hyperactivated PARP, hypersensitivity to PARP inhibitors, and are defective in HR [87]. In contrast, depletion of RECQ by itself does not lead to PARP hyperactivation or enhanced sensitivity to PARP inhibitor [57]. Moreover, RECQ -depleted cells are not compromised in their ability to repair I-SceI-induced DSB by homology directed repair [57]. In addition to HR, DSBs are repaired by NHEJ mediated by Ku70/80, the DNA-PKcs protein kinase, and the complex consisting of DNA ligase IV, XRCC4 and XLF [88]. Our more recent data indicates that RECQ and Ku70/80 co-bind a linear DNA and the DNA binding by Ku70/80 is modulated by the presence of RECQ [63]. Recent models of DSB repair propose that Ku binds DNA ends first and is subsequently released through the DNA end processing activities contributed by MRN complex,CtIP,. Ku70/80 inhibits EXO--mediated DSB resection in vivo [9 ] and DNA end resection of the forked duplex substrate in vitro [92]. The ability of RECQ to bind and unwind a Ku-bound forked DNA duplex relatively efficiently and its known interaction with EXO-suggests that RECQ may enable EXO-to overcome Ku inhibition and thereby modulate the pathway choice for DSB repair [92].
It is important to note that HR mechanisms involved in repairing classical two ended DSBs are distinct from those provoked by replication stress [93]. Notably, RECQ , along with RecQ4, is an integral component of replication complex in unperturbed dividing cells [94]. Association of RECQ with replication origins during normal replication is significantly enhanced when cells encounter replication stress [82,94]. Consistent with this, recombinant RECQ binds and unwinds model replication forks [ 4], and promotes strand exchange on stalled replication forks in vitro [8 ]. RECQ -depletion results in increased sensitivity to aphidicolin, diminished checkpoint activation in response to replication stress, and chromosomal instability [82].
Chromatin immunoprecipitation experiments revealed that RECQ is preferentially enriched at two major fragile sites, FRA3B and FRA 6D, where replication forks have stalled in vivo following aphidicolin treatment [82]. Common fragile sites are randomly distributed slow replicating genomic regions that are particularly vulnerable to replication stress and expressed as sitespecific gaps or breaks on metaphase chromosomes after partial inhibition of DNA synthesis [95]. Fragile sites often coincide with chromosomal breakpoints in tumors [95][96] and stalled replication forks at common fragile sites are believed to be a major cause of genomic instability [95,97]. Consistent with its demonstrated catalytic functions [65,8 ], recruitment of RECQ at fragile sites indicates that RECQ facilitates repair of stalled or collapsed replication forks and preserves genome integrity [82]. These results also implicate RECQ in mechanisms underlying common fragile site instability in cancer. Remarkably, RECQ is overexpressed in transformed cells [98] and in many clinical cancer samples compared to matched normal samples indicating potential target for cancer therapy [82,. Single nucleotide polymorphisms of RECQ have been associated with reduced survival in pancreatic cancer [ 0 -02]. RECQ expression is critical for the growth and proliferation of a variety of cancer cells [44,03]; this has also been demonstrated using xenograft models [ 04]. It is conceivable that cancer cells are overtly dependent upon RECQ activities to cope with replicationinduced DNA damage during rapid cell division; in normal cells, RECQ can act as a tumor suppressor by facilitating DNA repair and preventing mutations.

Summary and Outlook
Recent advances in structural analyses and identification of novel molecular interactions has provided a valuable foundation to explore unique and overlapping functions of RECQ . In particular, importance of RECQ in repair of DNA damage in the context of replication stress has become increasingly more apparent. DNA lesions induced by replication stress occur predominantly in early replicating and actively transcribed gene clusters [ 05]. Given the presence of RECQ at replication origins, it will be insightful to investigate whether RECQ has a role in preventing transcriptionassociated genetic instability. Molecular functions of RecQ and other DNA helicases in cancer are becoming more prominent [ 06]. Reported overexpression of RECQ in a variety of clinical cancer merits systematic investigation of clinicopathological correlation. While a disease association remains to be discovered, unique requirement of RECQ in suppressing genomic instability proposes that a defect in RECQ may be linked to cancer predisposition disorders that are distinct from known RecQ-diseases. A greater understanding of the molecular and cellular functions of RECQ is essential to establish its role in genome maintenance and explore its translational potential.