- Email: [email protected]
Escherichia coli RecQ helicase: A player in thymineless death
Mutation Research 577 (2005) 228–236
Review
Escherichia coli RecQ helicase: A player in thymineless death Hiroaki Nakayama ∗ Kyushu University (Emeritus), Fukuoka 812-8581, Japan Received 27 January 2005; received in revised form 25 February 2005; accepted 25 February 2005 Available online 25 May 2005
Abstract DNA helicases of the RecQ family are distributed among most organisms and are thought to play important roles in various aspects of DNA metabolism. The founding member of the family, RecQ of Escherichia coli, was identified in a study aimed at clarifying the mechanism of thymineless death, a phenomenon underlying the mechanism for the cytotoxicity of the anticancer drug 5-fluorouracil. The present article is concerned solely with E. coli RecQ and tries to offer an integrated picture of the past and present of its study. Finally a brief discussion is given on how RecQ is involved in thymineless death. © 2005 Elsevier B.V. All rights reserved. Keywords: Escherichia coli; RecQ; Thymineless death; DNA; Recombination
Contents 1. 2. 3. 4. 5. 6. 7.
∗
Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The discovery of RecQ helicase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular architecture and functional dissection of RecQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro activities of RecQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo roles of RecQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The TLD connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Present address: 3-27-5 Nishifukuma, Fukutsu, Fukuoka-ken 811-3219, Japan. Fax: +81 940 43 4099. E-mail address: [email protected].
0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.02.015
229 229 230 230 231 232 232 234 234 234
H. Nakayama / Mutation Research 577 (2005) 228–236
229
Prologue
1. Introduction
Having finished my Ph.D. thesis in medical microbiology at Kyushu University Medical School, I switched paths and entered the field of molecular biology in 1965. Soon after that, I became aware of the phenomenon of thymineless death (TLD) and the name of Philip C. Hanawalt (Phil) simultaneously through the benchmark paper by Maaløe and Hanawalt [1]. The work aroused my interest in the mechanism of TLD, but I did not commit myself immediately to the study of it. This was mainly because I was then quite fascinated by an enzyme, UV endonuclease from the bacterium Micrococcus lysodeikticus (now known as M. luteus), which was specific for UV-damaged DNA. Since the UvrABC-type enzyme was not yet known, UV endonuclease was an extremely attractive candidate for the nicking enzyme essential for the excision repair of such DNA. After achieving the partial purification and preliminary characterization of this enzyme, I wanted to do postdoctoral work in the laboratory of Phil, who was playing a major role in the development of excision repair studies. Fortunately, my wish was realized. I joined his lab in December of 1969, and started working on a problem in excision repair in Escherichia coli following a suggestion by Phil. Our aim was to test the possibility that the excision repair of UV-induced damage to the chromosomal DNA might occur preferentially in the region ahead of the replication fork compared to the region behind it (cowcatcher model). For this purpose, I used amino-acid starvation to align cells with respect to chromosome replication according to the Maaløe–Hanawalt procedure [1]. Somewhat to our disappointment, the results showed that the degree of repair was indistinguishable between these regions, thus arguing against the model. Unfortunately, this amusing name for the model coined by Phil did not appear in the publication [2] because the editor would not agree to its use for the reason that an abandoned model should not bear an attractive name. Anyway, this second encounter with the Maaløe–Hanawalt paper [1] revived the imprint in my mind. When Phil, who had by this stage published a series of additional papers on the subject [3–5], allowed me the freedom of choosing what to do next, I did not hesitate in sharing my interest in TLD with him. TLD has since been our common concern, which led to the discovery of RecQ helicase.
TLD is the loss of viability that takes place when growing cells of thymine-requiring Escherichia coli are transferred to a medium lacking thymine but otherwise sufficient for continued growth. This phenomenon was discovered by Cohen and Barner half a century ago [6] and attracted the attention of a number of researchers up to the 1970s. Then, the interest in it faded rather rapidly, with its precise mechanism left unsolved until now. These days, few biologists except those with gray hair know of it in spite of the fact that it is not specific to E. coli. In effect, it constitutes the underlying mechanism for the therapeutic action of 5-fluorouracil, one of the important anticancer drugs currently in use. In the early seventies, substantial evidence had already accumulated indicating that thymine starvation of E. coli cells caused DNA alterations. Thus, it was known to be mutagenic [7–9] and recombinogenic [10] and to elicit repair replication [4] and single-strand breaks [11]. Also, enhanced TLD accompanied by increased single-strand breaks in polA mutants began to be noticed [12]. We reasoned that if TLD was a consequence of some nonessential cellular process occurring in thymine-starved cells, mutations that block it would lead to resistance to the lethality. With the hope that such mutations might be more revealing than, or at least complementary to, those enhancing TLD, we tried to isolate such a mutant. The attempt was successful, and the characterization of the mutant obtained led to the discovery of the recQ gene and the gene product RecQ. Subsequent studies by a number of workers have revealed that homologs of RecQ, composing the RecQ family, are widely distributed across all three domains of life and that three of the five human homologs are causally linked with hereditary disorders. As a result, eukaryotic members of the RecQ family, especially yeast and human homologs, have become the main focus of intensive research, which is now yielding voluminous literature. The scope of the present article is confined essentially to those topics directly related to the prototype RecQ. After summarizing earlier work on the recQ gene and RecQ, I shall survey recent findings on structural features, in vitro activities, and in vivo functions of RecQ, and finally present some thoughts on its relationship with TLD.
230
H. Nakayama / Mutation Research 577 (2005) 228–236
2. The discovery of RecQ helicase The TLD-resistant mutant HN15 was obtained by subjecting mutagenized cells of the thymine-requiring E. coli strain AB2497 to selection by repeated thymine starvation. Initially, genetic analysis of the mutant was extremely cumbersome due to the absence of phenotype other than TLD resistance. Meanwhile, a study on TLD in recombination-deficient mutants revealed that recF mutants, known to be defective in the so-called RecF pathway of recombination [13], exhibited resistance to TLD [14]. This soon led to the finding that HN15 was also defective in the RecF pathway, hence the name recQ (Q for Kyushu University) for the gene responsible for both the TLD resistance and the recombination deficiency of the mutant [15]. Facilitated by this finding, genetic fine mapping followed by cloning of the recQ gene was achieved [15,16]. Also, it was confirmed by insertion mutagenesis that the disruption of the recQ gene actually conferred recombination deficiency as well as TLD resistance on the cell [16]. The primary structure of RecQ was determined from the nucleotide sequence of the cloned gene and analysis of the amino-terminal sequence of the purified protein [17,18]. Also, RecQ was shown to possess an ATPdependent DNA helicase activity, with the direction of duplex unwinding being 3 to 5 with respect to the DNA single strand to which the enzyme supposedly binds [18].
3. Molecular architecture and functional dissection of RecQ In the early stages of the study, the following features of RecQ were revealed by the survey of its aminoacid sequence. First of all, like most helicases, RecQ possesses seven conserved motifs (I, Ia, II, III, IV, V, VI) defined by Gorbalenya and Koonin [19]. Of these, motifs I and II are respectively the Walker’s NTP-binding motifs A and B. Incidentally, the mutation in strain HN15 (recQ1801, formerly recQ1) disrupts motif 1 by a serine to phenylalanine conversion (K. Nakayama, unpublished). Within the RecQ family, the seven motifs are located in well-conserved regions comprising 320–350 amino-acid residues (helicase region), and each motif serves as a signature for the family. Re-
cently, a hitherto unnoticed conserved sequence, motif 0, has been proposed [20]. Besides the helicase region, two additional regions have been identified downstream of it [21]. The proximal RecQ Ct (for C-terminal) region is apparently specific to the RecQ family and found in most family members, whereas the distal HRDC (for helicase and RNase D C-terminal) region is lacking in some members but shared by RNase D homologs and certain other helicases. The N-terminal and C-terminal regions of RecQ family proteins are highly variable with respect to both length and amino-acid sequence. In particular, the majority of eukaryotic homologs have long tails at one or both ends, some of which have been assigned functional roles. According to the results of limited proteolysis, RecQ has a two-domain structure consisting of a larger domain comprising the helicase and RecQ Ct regions and a smaller domain corresponding to the HRDC region [20]. Functional mapping using truncated versions of RecQ has shown that the large domain is sufficient to exhibit normal levels of the DNA helicase and DNAdependent ATPase activities in vitro but devoid of the ability to stably bind to DNA. Additionally, the removal of 20 N-terminal residues from RecQ, which disrupts motif 0, results in a drastic decrease in the helicase and ATPase activities but is without effect on the DNAbinding ability [20]. X-ray crystallographic analysis of the large domain has revealed that it comprises four subdomains, of which the two nearer the N-terminus make up the helicase region and the remaining two the RecQ Ct region [22]. Those subdomains composing the helicase region embrace a deep cleft, the walls of which are lined by the helicase motifs including motif 0. Interestingly, certain amino-acid residues constituting motif 0 participate in ATP binding. Provisional sites for interaction with single-stranded DNA are also assumed to be in the cleft. Of the two subdomains forming the RecQ Ct region, the upstream one is characterized by a structure involved in Zn2+ binding, which is likely to be formed by four cysteine residues. Although this Zn2+ -binding motif is not similar to the known zinc finger structures, the presence of Zn2+ has been confirmed biochemically. The other subdomain of the RecQ Ct region takes a helix-turn-helix structure called WH, known to act in DNA binding. It is surmised that a cleft formed between the two RecQ Ct subdo-
H. Nakayama / Mutation Research 577 (2005) 228–236
231
mains could serve as a binding site for double-stranded DNA.
4. In vitro activities of RecQ A salient feature of the kinetic property of RecQ helicase is a marked stimulation of the helixunwinding activity by single-stranded DNA-binding protein (SSB). Without SSB, stoichiometric amounts of RecQ are needed for detectable duplex unwinding, but the presence of SSB reduces the amount of RecQ required to catalytic levels [23,24]. This effect of SSB is likely to be due to its binding to the DNA single-strands, thereby stabilizing an unwound state of the substrate [23,24] and preventing futile binding of RecQ to existing single strands [24]. Also, the SSB dependence of RecQ may be a reflection of its presumed role in homologous recombination. Since the RecA-mediated strand exchange reactions are highly dependent on SSB [25], it would be reasonable to assume that RecQ and RecA are working in the close proximity of each other in an SSB-rich microenvironment within the cell. Recent studies have shown that RecQ, in conjunction with SSB, is capable of catalyzing a variety of reactions as depicted in Fig. 1. Thus, the concerted action of both the proteins promotes RecA-mediated homologous pairing of supercoiled closed circular DNA and linear duplex DNA to form joint molecules while it disrupts various duplex structures including joint molecules [26]. An intriguing implication of these results is that the two types of reactions may represent two opposite in vivo roles of RecQ: the initiation and reversal of recombination, the latter probably serving to counter aberrant recombination [26]. Furthermore, the cooperation of the two proteins not only unwinds a relaxed closed circular DNA substrate [27] but also stimulates topoisomerase III (Topo III) to change the linking number of closed circular DNA or catenate molecules of such DNA [27,28]. In these reactions involving Topo III, neither RecQ nor Topo III can be replaced with other helicases or topoisomerases respectively, indicating that the reactions specifically require the combination of RecQ and Topo III. This implies that the activity of this combination of proteins is not an in vitro artifact but may reflect a physiologically significant in vivo function. Also noteworthy is the finding that RecQ, even in the absence of SSB,
Fig. 1. Examples of RecQ-catalyzed reactions. SSB is contained in all reactions, whereas RecA or Topo III is included as indicated [26–28]. Simple unwinding reactions for partial or complete duplexes are not shown. In some figures, dsDNA is represented by thick single lines. Dots on ssDNA signify SSB.
is capable of unwinding a certain type of G–G paired tetraplex DNA, which can be a roadblock in DNA replication if present on the template strand [29]. Conceivably, all these in vitro findings have broad implications for in vivo roles of RecQ family helicases. There appears to be a conflict regarding the quaternary structure of RecQ. An oligomeric state was suggested on the basis of kinetic studies [24], which agrees with reports for certain eukaryotic homologs [30–32]. On the other hand, RecQ has been shown not only to exist as monomers in solution [18,33] but also to perform DNA unwinding reactions as such [33]. At present, this discrepancy remains an open question.
232
H. Nakayama / Mutation Research 577 (2005) 228–236
5. In vivo roles of RecQ The recQ mutation appears to exert opposite effects on the recombination of DNA depending on the circumstances. As is evident from the phenotype of recQ mutants, RecQ is required for the post-conjugational recombinant formation in recB recC sbcB mutants, but not in wild-type strains [15,16]. A plausible interpretation is that RecQ, in cooperation with RecJ exonuclease, would substitute for RecBCD protein in converting the end of the recombining duplex DNA to a 3 -tailed structure needed for the initiation of RecA-mediated strand exchange reactions. On the other hand, recQ mutations enhance a certain type of illegitimate recombination that is independent of RecA and requires short stretches of sequence homology [34,35]. In a model for this type of recombination [36], this effect of RecQ has been attributed to its ability to disrupt hydrogen-bonded recombination intermediates formed through the microhomologies. Overall, these findings are consistent with the in vitro observations that RecQ is capable of either forming or disrupting structures mimicking recombination intermediates [26]. In plasmid recombination involving plasmid dimers [37], the contribution of RecQ in a recB recC sbcB host is demonstrable for the recombination-mediated cyclization of linear dimers, but not for the intramolecular recombination of circular dimers. This apparent requirement of duplex ends may be accounted for by the same mechanism as in the case of conjugational recombination. In a recB recC sbcA strain, however, functional RecQ is a requirement for the recombination of circular dimers, but not of linear dimers. Although no satisfactory explanation is presently available for this apparent contradiction, it must be noted that the recombination is RecA dependent in the former strain while RecE dependent in the latter. Additionally, like some other RecF pathway proteins, RecQ seems to be required also for the UV survival of recB recC sbcB mutants, which may possibly involve recombination [15,16]. This appears to be consistent with the fact that the recQ gene expression is subject to an SOS-like, damage-inducible mode of regulation [17]. Recently, RecQ has been demonstrated to play a role in chromosome replication as well [38–41]. In fact, many recombination proteins other than RecQ also participate in replication as factors required for restoring
the integrity of arrested replication forks [42], raising a question about the raisons d’`etre of those proteins [43]. Two distinct roles of RecQ in replication may be conceived. One is a role in processing arrested replication forks. When a replication fork is stalled by a block such as a UV-induced photoproduct, the nascent lagging-strand DNA is degraded by the combined action of RecQ helicase and RecJ exonuclease in a manner controlled by RecF, RecO and RecR (RecFOR) proteins [38–40]. This results in an extended singlestranded region on the lagging strand template, which would facilitate the loading of RecA in the presence of RecFOR [40]. It is argued that the RecA loading not only provides direct protection to stalled replication forks [38–40] but also activates RecA to generate the SOS signal [41]. Notably, however, neither recQ mutations nor recJ mutations make the cell UV sensitive by themselves [15,44]. Hence, the physiological significance of the RecQJ-dependent nascent DNA degradation appears to remain an open question. The other is a role as a roadblock remover, i.e., the unwinding of secondary structures on the template strand that block replication. In this connection, an interesting observation is that RecQ allows DNA replication to pass through a hairpin structure on the template strand [45]. G4 tetraplex DNA may also be unwound by RecQ in vivo since such activity has been demonstrated in vitro [29]. Finally, in view of the in vitro synergy between RecQ and Topo III [27,28], its biological significance must be considered. In effect, in vivo functional interactions between eukaryotic RecQ and Topo III are well documented (for example, see ref. [46]). In this context, it is interesting to note that E. coli cells defective in both RecQ and Topo III are phenotypically similar to those doubly deficient in Topo I and Topo III, which are characterized by a chromosome segregation anomaly suppressible by recA mutations [47]. Obviously, this problem needs further studies.
6. The TLD connection Now I return to the origin of RecQ studies and ask the question of how RecQ deficiency leads to TLD resistance. Here, I shall limit the discussion to those points directly related to the hypothetical role played
H. Nakayama / Mutation Research 577 (2005) 228–236
233
Fig. 2. Schematic representation of tentative models for TLD. The bulk model assumes many events such as this to occur along the length of sister chromatids. The gene products shown in parentheses are potential mediators in respective steps. Arrowheads signify the 3 -termini of DNA strands.
by RecQ, leaving more extensive treatment of the topic for future publication. As mentioned earlier, the primary assumption is that TLD results from DNA changes brought about by thymine starvation, where RecQ takes part actively in the formation of eventual lethal damage to DNA. Conceptually, there should be two possibilities as to the potential site of lethal damage (Fig. 2). One possibility is that changes in the bulk DNA elicited by thymine deficiency [4,12,48] may lead to irreparable lethal damage (the bulk model), whereas the other is that replication forks arrested by thymine deprivation will generate an increased risk of potentially lethal fork demise (the fork model). Needless to say, these possibilities are not mutually exclusive; perhaps, both contribute to the lethality to different degrees. In considering these models, an important key to a solution would be TLD sensitivities of rec mutants. Like recQ and recF mutants, recJ and recO mutants are also TLD resistant [49]. recA mutants are as TLD sensitive as wild-type strains while recB and recC mutants are TLD hypersensitive [14]. Of particular interest is
the TLD resistance of recF and recO mutants. As mentioned above, RecF and RecO, along with RecR, are known to be required for loading RecA on SSB-coated single-stranded DNA [50–52]. It is important to note that the RecFOR-dependent RecA loading not only affords protection to arrested replication forks [40] but also precedes the RecA-dependent strand exchange reactions in homologous recombination [52]. In thymine-starved cells, the cellular DNA contains portions that exhibit an abnormal electrophoretic behavior and are rich in long single-stranded regions and Y or X-shaped structures with single-stranded arms [53]. The amount of such DNA is smaller in recA, recF, recJ, recO, and recQ mutants than in the wild-type strain while it is markedly increased in a recB recC mutant [53]. The following interpretation of these observations is an extension of the previously proposed idea [12] that single-strand breaks occasionally formed in the bulk of chromosomal DNA would remain unrepaired in the absence of thymine. Thus, in thymine-deprived cells, those persisting single-strand interruptions would develop into large gaps and lead
234
H. Nakayama / Mutation Research 577 (2005) 228–236
to excessive recombinational events between the sister chromatids in a manner dependent on RecA, RecF, RecJ RecO and RecQ. Those events would result in a number of aberrant recombination intermediates with long single-stranded regions, which could eventually make the restoration of the chromosome impossible (Fig. 2). It is notable that the TLD resistance parallels the repressed formation of the abnormal DNA in all of the mutants tested except for recA. Clearly, the bulk model can not be substantiated unless this recA anomaly is explained. (The mechanism for increase in the abnormal DNA formation in the recB recC mutant is presently not clear.) Little is known about the replication fork arrest by thymine deprivation. In the case of UV irradiation, deficiency in any one of the RecFOR proteins renders the cell hypersensitive to UV [13,54,55], probably due to the cell’s inability to protect arrested replication forks [40]. By analogy, if inadequate protection of stalled replication forks should be mainly responsible for TLD, the recF and recO mutants are expected to be hypersensitive rather than resistant to TLD (Fig. 2). This appears to make the fork model less likely. However, it is conceivable that the fork failure may account for a fraction of TLD or, to be more specific, the residual TLD sensitivity of recF, recJ, recO, and recQ mutants [14,16,49]. Another corollary of this idea is that the above-mentioned recA conundrum may reflect the contribution of replication fork disruption. Thus, in recA mutants, replication forks stalled by thymine deficiency may be disrupted with increased frequencies, and the resulting increase in lethality would counter the repressive effect of RecA deficiency on the recombinationdependent lethal events involving the bulk DNA. This may account for the apparent indifference of the recA mutation to TLD [14].
7. Coda TLD is a ubiquitous phenomenon. It occurs in most, if not all, cells; even the monstrous bacterium Deinococcus radiodurans with an enormous capacity to repair DNA double-strand breaks is not free from it [56]. Our attempt to learn more about it culminated in the identification and characterization of the recQ gene and the gene product RecQ. Although the discovery of RecQ did not lead us directly to the final solution, it
certainly marked a significant step toward understanding TLD. It is hoped that a perfect answer to the puzzle could be obtained by developing the ideas discussed above.
Acknowledgements I thank K. Umezu and K. Nakayama for critical reading of the manuscript. K. Nakayama also kindly permitted me to cite his unpublished results. Comments from I. Grant and M. Inoue on the English of an earlier version of the manuscript are acknowledged with appreciation.
References [1] O. Maaløe, P.C. Hanawalt, Thymine deficiency and the normal DNA replication cycle. I, J. Mol. Biol. 3 (1961) 144–155. [2] H. Nakayama, A. Pratt, P. Hanawalt, Ultraviolet-induced repair synthesis in the duplicated and non-duplicated regions of the Escherichia coli chromosome, J. Mol. Biol. 70 (1972) 281–289. [3] P.C. Hanawalt, Involvement of synthesis of RNA in thymineless death, Nature 198 (1963) 286. [4] C. Pauling, P. Hanawalt, Nonconservative DNA replication in bacteria after thymine starvation, Proc. Natl. Acad. Sci. U.S.A. 54 (1965) 1728–1735. [5] D.W. Smith, P.C. Hanawalt, Macromolecular synthesis and thymineless death in Mycoplasma laidlawii B, J. Bacteriol. 96 (1968) 2066–2076. [6] S.S. Cohen, H.D. Barner, Studies on unbalanced growth in E. coli, Proc. Natl. Acad. Sci. U.S.A. 40 (1954) 885–893. [7] C.A. Coughlin, E.A. Adelberg, Bacterial mutation induced by thymine starvation, Nature 178 (1956) 531–532. [8] R. Weinberg, A.B. Latham, Apparent mutagenic effect of thymine deficiency for a thymine-requiring strain of Escherichia coli, J. Bacteriol. 72 (1956) 570–572. [9] D. Kanazir, The apparent mutagenicity of thymine deficiency, Biochim. Biophys. Acta 30 (1958) 20–23. [10] J. Gallant, T. Spottswood, Measurement of the stability of the repressor of alkaline phosphatase synthesis in E. coli, Proc. Natl. Acad. Sci. U.S.A. 52 (1964) 1591–1598. [11] D. Freifelder, Single-strand breaks in bacterial DNA associated with thymine starvation, J. Mol. Biol. 45 (1969) 1–7. [12] H. Nakayama, P. Hanawalt, Sedimentation analysis of deoxyribonucleic acid from thymine-starved Escherichia coli, J. Bacteriol. 121 (1975) 537–547. [13] Z.-I. Horii, A.J. Clark, Genetic analysis of the RecF pathway of genetic recombination in Escherichia coli K12: isolation and characterization of mutants, J. Mol. Biol. 80 (1973) 327–344. [14] H. Nakayama, K. Nakayama, R. Nakayama, Y. Nakayama, Recombination-deficient mutations and thymineless death in Escherichia coli K12: reciprocal effects of recBC and recF and
H. Nakayama / Mutation Research 577 (2005) 228–236
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26]
[27]
[28]
[29]
[30]
indifference of recA mutations, Can. J. Microbiol. 28 (1982) 425–430. H. Nakayama, K. Nakayama, R. Nakayama, N. Irino, Y. Nakayama, P.C. Hanawalt, Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway, Mol. Gen. Genet. 195 (1984) 474–480. K. Nakayama, N. Irino, H. Nakayama, The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants, Mol. Gen. Genet. 200 (1985) 266–271. N. Irino, K. Nakayama, H. Nakayama, The recQ gene of Escherichia coli K12: primary structure and evidence for SOS regulation, Mol. Gen. Genet. 205 (1986) 298–304. K. Umezu, K. Nakayama, H. Nakayama, Escherichia coli RecQ protein is a DNA helicase, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 5363–5367. A.E. Gorbalenya, E.V. Koonin, Helicases: amino acid sequence comparisons and structure-function relationships, Curr. Opin. Struct. Biol. 3 (1993) 419–429. D.A. Bernstein, J.L. Keck, Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family, Nucleic Acids Res. 31 (2003) 2778–2785. V. Morozov, A.R. Mushegian, E.V. Koonin, P. Bork, A putative nucleic acid-binding domain in Bloom’s and Werner’s syndrome helicases, Trends Biochem. Sci. 22 (1997) 417–418. D.A. Bernstein, M.C. Zittel, J.L. Keck, High-resolution structure of the E. coli RecQ helicase catalytic core, EMBO J. 22 (2003) 4910–4921. K. Umezu, H. Nakayama, RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein, J. Mol. Biol. 230 (1993) 1145–1150. F.G. Harmon, S.C. Kowalczykowski, Biochemical characterization of the DNA helicase activity of Escherichia coli RecQ helicase, J. Biol. Chem. 276 (2001) 232–243. R.R. Meyer, P.S. Laine, The single-stranded DNA-binding protein of Escherichia coli, Microbiol. Rev. 54 (1990) 342–380. F.G. Harmon, S.C. Kowalczykowski, RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination, Genes Dev. 12 (1998) 1134–1144. F.G. Harmon, R.J. DiGate, S.C. Kowalczykowski, RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination, Mol. Cell 3 (1999) 611–620. F.G. Harmon, J.P. Brockman, S.C. Kowalczykowski, RecQ helicase stimulates both DNA catenation and changes in DNA topology by topoisomerase III, J. Biol. Chem. 278 (2003) 42668–42678. A.S. Kamath-Loeb, L.A. Loeb, E. Johansson, P.M.J. Burgers, M. Fry, Interactions between the Werner syndrome helicase and DNA polymerase ␦ specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence, J. Biol. Chem. 276 (2001) 16439–16446. J.K. Karow, R.H. Newman, P.S. Freemont, I.D. Hickson, Oligomeric ring structure of the Bloom’s syndrome helicase, Current Biol. 9 (1999) 597–600.
235
[31] S. Huang, S. Beresten, B. Li, J. Oshima, N.A. Ellis, J. Campisi, Characterization of the human and mouse WRN 3 → 5 exonuclease, Nucleic Acids Res. 28 (2000) 2396–2405. [32] S. Cui, R. Klima, A. Ochem, D. Arosio, A. Falaschi, A. Vindigni, Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A, J. Biol. Chem. 278 (2003) 1424– 1432. [33] H.Q. Xu, E. Deprez, A.H. Zhang, P. Tauc, M.M. Ladjimi, J.C. Brochon, C. Auclair, X.G. Xi, The Escherichia coli RecQ helicase functions as a monomer, J. Biol. Chem. 278 (2003) 34925–34933. [34] K. Hanada, T. Ukita, Y. Kohno, K. Saito, J. Kato, H. Ikeda, RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3860–3865. [35] K. Shiraishi, K. Hanada, Y. Iwakura, H. Ikeda, Roles of RecJ, RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli, J. Bacteriol. 184 (2002) 4715–4721. [36] H. Ikeda, Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli, in: S. Ebashi (Ed.), Advances of Biophysics, vol. 38, Japan Scientific Societies Press, Elsevier, Tokyo, Limerick, 2004, pp. 3–20. [37] C. Luisi-DeLuca, S.T. Lovett, R.D. Kolodner, Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants, Genetics 122 (1989) 269–278. [38] J. Courcelle, P.C. Hanawalt, RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli, Mol. Gen. Genet. 262 (1999) 543–551. [39] J. Courcelle, J.R. Donaldson, K.-H. Chow, C.T. Courcelle, DNA damage-induced replication fork regression and processing in Escherichia coli, Science 299 (2003) 1064–1067. [40] K.-H. Chow, J. Courcelle, RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli, J. Biol. Chem. 279 (2004) 3492–3496. [41] T. Hishida, Y.-W. Han, T. Shibata, Y. Kubota, Y. Ishino, H. Iwasaki, H. Shinagawa, Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks, Genes Dev. 18 (2004) 1886–1897. [42] J. Courcelle, P.C. Hanawalt, RecA-dependent recovery of arrested DNA replication forks, Annu. Rev. Genet. 37 (2003) 611–646. [43] J. Courcelle, A.K. Ganesan, P.C. Hanawalt, Therefore, what are recombination proteins there for? BioEssays 23 (2001) 463–470. [44] S.T. Lovett, A.J. Clark, Genetic analysis of the recJ gene of Escherichia coli K-12, J. Bacteriol. 157 (1984) 190–196. [45] G.A. Cromie, C.B. Millar, K.H. Schmidt, D.R.F. Leach, Palindromes as substrates for multiple pathways of recombination in Escherichia coli, Genetics 154 (2000) 513–522. [46] S. Gangloff, J.P. McDonald, C. Bendixen, L. Arthur, R. Rothstein, The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase, Mol. Cell. Biol. 14 (1994) 8391–8398.
236
H. Nakayama / Mutation Research 577 (2005) 228–236
[47] Q. Zhu, P. Pongpech, R.J. DiGate, Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 9766–9771. [48] C.E. Deutch, C. Pauling, Thymineless mutagenesis in Escherichia coli, J. Bacteriol. 119 (1974) 861–867. [49] K. Nakayama, S. Shiota, H. Nakayama, Thymineless death in Escherichia coli mutants deficient in the RecF recombination pathway, Can. J. Microbiol. 34 (1988) 905–907. [50] K. Umezu, N.-W. Chi, R.D. Kolodner, Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 3875–3879. [51] K. Umezu, R.D. Kolodner, Protein interactions in genetic recombination in Escherichia coli, J. Biol. Chem. 269 (1994) 30005–30013. [52] K. Morimatsu, S.C. Kowalczykowski, RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand ex-
[53]
[54]
[55]
[56]
change: a universal step in recombinational repair, Mol. Cell 11 (2003) 1337–1347. K. Nakayama, K. Kusano, N. Irino, H. Nakayama, Thymine starvation-induced structural changes in Escherichia coli DNA. Detection by pulsed field gel electrophoresis and evidence for involvement of homologous recombination, J. Mol. Biol. 243 (1994) 611–620. R. Kolodner, R.A. Fishel, M. Howard, Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli, J. Bacteriol. 163 (1985) 1060–1066. A.A. Mahdi, R.G. Lloyd, Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair, Mol. Gen. Genet. 216 (1989) 503–510. J.G. Little, P.C. Hanawalt, Thymineless death and ultraviolet sensitivity in Micrococcus radiodurans, J. Bacteriol. 113 (1973) 233–240.
Review
Escherichia coli RecQ helicase: A player in thymineless death Hiroaki Nakayama ∗ Kyushu University (Emeritus), Fukuoka 812-8581, Japan Received 27 January 2005; received in revised form 25 February 2005; accepted 25 February 2005 Available online 25 May 2005
Abstract DNA helicases of the RecQ family are distributed among most organisms and are thought to play important roles in various aspects of DNA metabolism. The founding member of the family, RecQ of Escherichia coli, was identified in a study aimed at clarifying the mechanism of thymineless death, a phenomenon underlying the mechanism for the cytotoxicity of the anticancer drug 5-fluorouracil. The present article is concerned solely with E. coli RecQ and tries to offer an integrated picture of the past and present of its study. Finally a brief discussion is given on how RecQ is involved in thymineless death. © 2005 Elsevier B.V. All rights reserved. Keywords: Escherichia coli; RecQ; Thymineless death; DNA; Recombination
Contents 1. 2. 3. 4. 5. 6. 7.
∗
Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The discovery of RecQ helicase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular architecture and functional dissection of RecQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro activities of RecQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo roles of RecQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The TLD connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Present address: 3-27-5 Nishifukuma, Fukutsu, Fukuoka-ken 811-3219, Japan. Fax: +81 940 43 4099. E-mail address: [email protected].
0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.02.015
229 229 230 230 231 232 232 234 234 234
H. Nakayama / Mutation Research 577 (2005) 228–236
229
Prologue
1. Introduction
Having finished my Ph.D. thesis in medical microbiology at Kyushu University Medical School, I switched paths and entered the field of molecular biology in 1965. Soon after that, I became aware of the phenomenon of thymineless death (TLD) and the name of Philip C. Hanawalt (Phil) simultaneously through the benchmark paper by Maaløe and Hanawalt [1]. The work aroused my interest in the mechanism of TLD, but I did not commit myself immediately to the study of it. This was mainly because I was then quite fascinated by an enzyme, UV endonuclease from the bacterium Micrococcus lysodeikticus (now known as M. luteus), which was specific for UV-damaged DNA. Since the UvrABC-type enzyme was not yet known, UV endonuclease was an extremely attractive candidate for the nicking enzyme essential for the excision repair of such DNA. After achieving the partial purification and preliminary characterization of this enzyme, I wanted to do postdoctoral work in the laboratory of Phil, who was playing a major role in the development of excision repair studies. Fortunately, my wish was realized. I joined his lab in December of 1969, and started working on a problem in excision repair in Escherichia coli following a suggestion by Phil. Our aim was to test the possibility that the excision repair of UV-induced damage to the chromosomal DNA might occur preferentially in the region ahead of the replication fork compared to the region behind it (cowcatcher model). For this purpose, I used amino-acid starvation to align cells with respect to chromosome replication according to the Maaløe–Hanawalt procedure [1]. Somewhat to our disappointment, the results showed that the degree of repair was indistinguishable between these regions, thus arguing against the model. Unfortunately, this amusing name for the model coined by Phil did not appear in the publication [2] because the editor would not agree to its use for the reason that an abandoned model should not bear an attractive name. Anyway, this second encounter with the Maaløe–Hanawalt paper [1] revived the imprint in my mind. When Phil, who had by this stage published a series of additional papers on the subject [3–5], allowed me the freedom of choosing what to do next, I did not hesitate in sharing my interest in TLD with him. TLD has since been our common concern, which led to the discovery of RecQ helicase.
TLD is the loss of viability that takes place when growing cells of thymine-requiring Escherichia coli are transferred to a medium lacking thymine but otherwise sufficient for continued growth. This phenomenon was discovered by Cohen and Barner half a century ago [6] and attracted the attention of a number of researchers up to the 1970s. Then, the interest in it faded rather rapidly, with its precise mechanism left unsolved until now. These days, few biologists except those with gray hair know of it in spite of the fact that it is not specific to E. coli. In effect, it constitutes the underlying mechanism for the therapeutic action of 5-fluorouracil, one of the important anticancer drugs currently in use. In the early seventies, substantial evidence had already accumulated indicating that thymine starvation of E. coli cells caused DNA alterations. Thus, it was known to be mutagenic [7–9] and recombinogenic [10] and to elicit repair replication [4] and single-strand breaks [11]. Also, enhanced TLD accompanied by increased single-strand breaks in polA mutants began to be noticed [12]. We reasoned that if TLD was a consequence of some nonessential cellular process occurring in thymine-starved cells, mutations that block it would lead to resistance to the lethality. With the hope that such mutations might be more revealing than, or at least complementary to, those enhancing TLD, we tried to isolate such a mutant. The attempt was successful, and the characterization of the mutant obtained led to the discovery of the recQ gene and the gene product RecQ. Subsequent studies by a number of workers have revealed that homologs of RecQ, composing the RecQ family, are widely distributed across all three domains of life and that three of the five human homologs are causally linked with hereditary disorders. As a result, eukaryotic members of the RecQ family, especially yeast and human homologs, have become the main focus of intensive research, which is now yielding voluminous literature. The scope of the present article is confined essentially to those topics directly related to the prototype RecQ. After summarizing earlier work on the recQ gene and RecQ, I shall survey recent findings on structural features, in vitro activities, and in vivo functions of RecQ, and finally present some thoughts on its relationship with TLD.
230
H. Nakayama / Mutation Research 577 (2005) 228–236
2. The discovery of RecQ helicase The TLD-resistant mutant HN15 was obtained by subjecting mutagenized cells of the thymine-requiring E. coli strain AB2497 to selection by repeated thymine starvation. Initially, genetic analysis of the mutant was extremely cumbersome due to the absence of phenotype other than TLD resistance. Meanwhile, a study on TLD in recombination-deficient mutants revealed that recF mutants, known to be defective in the so-called RecF pathway of recombination [13], exhibited resistance to TLD [14]. This soon led to the finding that HN15 was also defective in the RecF pathway, hence the name recQ (Q for Kyushu University) for the gene responsible for both the TLD resistance and the recombination deficiency of the mutant [15]. Facilitated by this finding, genetic fine mapping followed by cloning of the recQ gene was achieved [15,16]. Also, it was confirmed by insertion mutagenesis that the disruption of the recQ gene actually conferred recombination deficiency as well as TLD resistance on the cell [16]. The primary structure of RecQ was determined from the nucleotide sequence of the cloned gene and analysis of the amino-terminal sequence of the purified protein [17,18]. Also, RecQ was shown to possess an ATPdependent DNA helicase activity, with the direction of duplex unwinding being 3 to 5 with respect to the DNA single strand to which the enzyme supposedly binds [18].
3. Molecular architecture and functional dissection of RecQ In the early stages of the study, the following features of RecQ were revealed by the survey of its aminoacid sequence. First of all, like most helicases, RecQ possesses seven conserved motifs (I, Ia, II, III, IV, V, VI) defined by Gorbalenya and Koonin [19]. Of these, motifs I and II are respectively the Walker’s NTP-binding motifs A and B. Incidentally, the mutation in strain HN15 (recQ1801, formerly recQ1) disrupts motif 1 by a serine to phenylalanine conversion (K. Nakayama, unpublished). Within the RecQ family, the seven motifs are located in well-conserved regions comprising 320–350 amino-acid residues (helicase region), and each motif serves as a signature for the family. Re-
cently, a hitherto unnoticed conserved sequence, motif 0, has been proposed [20]. Besides the helicase region, two additional regions have been identified downstream of it [21]. The proximal RecQ Ct (for C-terminal) region is apparently specific to the RecQ family and found in most family members, whereas the distal HRDC (for helicase and RNase D C-terminal) region is lacking in some members but shared by RNase D homologs and certain other helicases. The N-terminal and C-terminal regions of RecQ family proteins are highly variable with respect to both length and amino-acid sequence. In particular, the majority of eukaryotic homologs have long tails at one or both ends, some of which have been assigned functional roles. According to the results of limited proteolysis, RecQ has a two-domain structure consisting of a larger domain comprising the helicase and RecQ Ct regions and a smaller domain corresponding to the HRDC region [20]. Functional mapping using truncated versions of RecQ has shown that the large domain is sufficient to exhibit normal levels of the DNA helicase and DNAdependent ATPase activities in vitro but devoid of the ability to stably bind to DNA. Additionally, the removal of 20 N-terminal residues from RecQ, which disrupts motif 0, results in a drastic decrease in the helicase and ATPase activities but is without effect on the DNAbinding ability [20]. X-ray crystallographic analysis of the large domain has revealed that it comprises four subdomains, of which the two nearer the N-terminus make up the helicase region and the remaining two the RecQ Ct region [22]. Those subdomains composing the helicase region embrace a deep cleft, the walls of which are lined by the helicase motifs including motif 0. Interestingly, certain amino-acid residues constituting motif 0 participate in ATP binding. Provisional sites for interaction with single-stranded DNA are also assumed to be in the cleft. Of the two subdomains forming the RecQ Ct region, the upstream one is characterized by a structure involved in Zn2+ binding, which is likely to be formed by four cysteine residues. Although this Zn2+ -binding motif is not similar to the known zinc finger structures, the presence of Zn2+ has been confirmed biochemically. The other subdomain of the RecQ Ct region takes a helix-turn-helix structure called WH, known to act in DNA binding. It is surmised that a cleft formed between the two RecQ Ct subdo-
H. Nakayama / Mutation Research 577 (2005) 228–236
231
mains could serve as a binding site for double-stranded DNA.
4. In vitro activities of RecQ A salient feature of the kinetic property of RecQ helicase is a marked stimulation of the helixunwinding activity by single-stranded DNA-binding protein (SSB). Without SSB, stoichiometric amounts of RecQ are needed for detectable duplex unwinding, but the presence of SSB reduces the amount of RecQ required to catalytic levels [23,24]. This effect of SSB is likely to be due to its binding to the DNA single-strands, thereby stabilizing an unwound state of the substrate [23,24] and preventing futile binding of RecQ to existing single strands [24]. Also, the SSB dependence of RecQ may be a reflection of its presumed role in homologous recombination. Since the RecA-mediated strand exchange reactions are highly dependent on SSB [25], it would be reasonable to assume that RecQ and RecA are working in the close proximity of each other in an SSB-rich microenvironment within the cell. Recent studies have shown that RecQ, in conjunction with SSB, is capable of catalyzing a variety of reactions as depicted in Fig. 1. Thus, the concerted action of both the proteins promotes RecA-mediated homologous pairing of supercoiled closed circular DNA and linear duplex DNA to form joint molecules while it disrupts various duplex structures including joint molecules [26]. An intriguing implication of these results is that the two types of reactions may represent two opposite in vivo roles of RecQ: the initiation and reversal of recombination, the latter probably serving to counter aberrant recombination [26]. Furthermore, the cooperation of the two proteins not only unwinds a relaxed closed circular DNA substrate [27] but also stimulates topoisomerase III (Topo III) to change the linking number of closed circular DNA or catenate molecules of such DNA [27,28]. In these reactions involving Topo III, neither RecQ nor Topo III can be replaced with other helicases or topoisomerases respectively, indicating that the reactions specifically require the combination of RecQ and Topo III. This implies that the activity of this combination of proteins is not an in vitro artifact but may reflect a physiologically significant in vivo function. Also noteworthy is the finding that RecQ, even in the absence of SSB,
Fig. 1. Examples of RecQ-catalyzed reactions. SSB is contained in all reactions, whereas RecA or Topo III is included as indicated [26–28]. Simple unwinding reactions for partial or complete duplexes are not shown. In some figures, dsDNA is represented by thick single lines. Dots on ssDNA signify SSB.
is capable of unwinding a certain type of G–G paired tetraplex DNA, which can be a roadblock in DNA replication if present on the template strand [29]. Conceivably, all these in vitro findings have broad implications for in vivo roles of RecQ family helicases. There appears to be a conflict regarding the quaternary structure of RecQ. An oligomeric state was suggested on the basis of kinetic studies [24], which agrees with reports for certain eukaryotic homologs [30–32]. On the other hand, RecQ has been shown not only to exist as monomers in solution [18,33] but also to perform DNA unwinding reactions as such [33]. At present, this discrepancy remains an open question.
232
H. Nakayama / Mutation Research 577 (2005) 228–236
5. In vivo roles of RecQ The recQ mutation appears to exert opposite effects on the recombination of DNA depending on the circumstances. As is evident from the phenotype of recQ mutants, RecQ is required for the post-conjugational recombinant formation in recB recC sbcB mutants, but not in wild-type strains [15,16]. A plausible interpretation is that RecQ, in cooperation with RecJ exonuclease, would substitute for RecBCD protein in converting the end of the recombining duplex DNA to a 3 -tailed structure needed for the initiation of RecA-mediated strand exchange reactions. On the other hand, recQ mutations enhance a certain type of illegitimate recombination that is independent of RecA and requires short stretches of sequence homology [34,35]. In a model for this type of recombination [36], this effect of RecQ has been attributed to its ability to disrupt hydrogen-bonded recombination intermediates formed through the microhomologies. Overall, these findings are consistent with the in vitro observations that RecQ is capable of either forming or disrupting structures mimicking recombination intermediates [26]. In plasmid recombination involving plasmid dimers [37], the contribution of RecQ in a recB recC sbcB host is demonstrable for the recombination-mediated cyclization of linear dimers, but not for the intramolecular recombination of circular dimers. This apparent requirement of duplex ends may be accounted for by the same mechanism as in the case of conjugational recombination. In a recB recC sbcA strain, however, functional RecQ is a requirement for the recombination of circular dimers, but not of linear dimers. Although no satisfactory explanation is presently available for this apparent contradiction, it must be noted that the recombination is RecA dependent in the former strain while RecE dependent in the latter. Additionally, like some other RecF pathway proteins, RecQ seems to be required also for the UV survival of recB recC sbcB mutants, which may possibly involve recombination [15,16]. This appears to be consistent with the fact that the recQ gene expression is subject to an SOS-like, damage-inducible mode of regulation [17]. Recently, RecQ has been demonstrated to play a role in chromosome replication as well [38–41]. In fact, many recombination proteins other than RecQ also participate in replication as factors required for restoring
the integrity of arrested replication forks [42], raising a question about the raisons d’`etre of those proteins [43]. Two distinct roles of RecQ in replication may be conceived. One is a role in processing arrested replication forks. When a replication fork is stalled by a block such as a UV-induced photoproduct, the nascent lagging-strand DNA is degraded by the combined action of RecQ helicase and RecJ exonuclease in a manner controlled by RecF, RecO and RecR (RecFOR) proteins [38–40]. This results in an extended singlestranded region on the lagging strand template, which would facilitate the loading of RecA in the presence of RecFOR [40]. It is argued that the RecA loading not only provides direct protection to stalled replication forks [38–40] but also activates RecA to generate the SOS signal [41]. Notably, however, neither recQ mutations nor recJ mutations make the cell UV sensitive by themselves [15,44]. Hence, the physiological significance of the RecQJ-dependent nascent DNA degradation appears to remain an open question. The other is a role as a roadblock remover, i.e., the unwinding of secondary structures on the template strand that block replication. In this connection, an interesting observation is that RecQ allows DNA replication to pass through a hairpin structure on the template strand [45]. G4 tetraplex DNA may also be unwound by RecQ in vivo since such activity has been demonstrated in vitro [29]. Finally, in view of the in vitro synergy between RecQ and Topo III [27,28], its biological significance must be considered. In effect, in vivo functional interactions between eukaryotic RecQ and Topo III are well documented (for example, see ref. [46]). In this context, it is interesting to note that E. coli cells defective in both RecQ and Topo III are phenotypically similar to those doubly deficient in Topo I and Topo III, which are characterized by a chromosome segregation anomaly suppressible by recA mutations [47]. Obviously, this problem needs further studies.
6. The TLD connection Now I return to the origin of RecQ studies and ask the question of how RecQ deficiency leads to TLD resistance. Here, I shall limit the discussion to those points directly related to the hypothetical role played
H. Nakayama / Mutation Research 577 (2005) 228–236
233
Fig. 2. Schematic representation of tentative models for TLD. The bulk model assumes many events such as this to occur along the length of sister chromatids. The gene products shown in parentheses are potential mediators in respective steps. Arrowheads signify the 3 -termini of DNA strands.
by RecQ, leaving more extensive treatment of the topic for future publication. As mentioned earlier, the primary assumption is that TLD results from DNA changes brought about by thymine starvation, where RecQ takes part actively in the formation of eventual lethal damage to DNA. Conceptually, there should be two possibilities as to the potential site of lethal damage (Fig. 2). One possibility is that changes in the bulk DNA elicited by thymine deficiency [4,12,48] may lead to irreparable lethal damage (the bulk model), whereas the other is that replication forks arrested by thymine deprivation will generate an increased risk of potentially lethal fork demise (the fork model). Needless to say, these possibilities are not mutually exclusive; perhaps, both contribute to the lethality to different degrees. In considering these models, an important key to a solution would be TLD sensitivities of rec mutants. Like recQ and recF mutants, recJ and recO mutants are also TLD resistant [49]. recA mutants are as TLD sensitive as wild-type strains while recB and recC mutants are TLD hypersensitive [14]. Of particular interest is
the TLD resistance of recF and recO mutants. As mentioned above, RecF and RecO, along with RecR, are known to be required for loading RecA on SSB-coated single-stranded DNA [50–52]. It is important to note that the RecFOR-dependent RecA loading not only affords protection to arrested replication forks [40] but also precedes the RecA-dependent strand exchange reactions in homologous recombination [52]. In thymine-starved cells, the cellular DNA contains portions that exhibit an abnormal electrophoretic behavior and are rich in long single-stranded regions and Y or X-shaped structures with single-stranded arms [53]. The amount of such DNA is smaller in recA, recF, recJ, recO, and recQ mutants than in the wild-type strain while it is markedly increased in a recB recC mutant [53]. The following interpretation of these observations is an extension of the previously proposed idea [12] that single-strand breaks occasionally formed in the bulk of chromosomal DNA would remain unrepaired in the absence of thymine. Thus, in thymine-deprived cells, those persisting single-strand interruptions would develop into large gaps and lead
234
H. Nakayama / Mutation Research 577 (2005) 228–236
to excessive recombinational events between the sister chromatids in a manner dependent on RecA, RecF, RecJ RecO and RecQ. Those events would result in a number of aberrant recombination intermediates with long single-stranded regions, which could eventually make the restoration of the chromosome impossible (Fig. 2). It is notable that the TLD resistance parallels the repressed formation of the abnormal DNA in all of the mutants tested except for recA. Clearly, the bulk model can not be substantiated unless this recA anomaly is explained. (The mechanism for increase in the abnormal DNA formation in the recB recC mutant is presently not clear.) Little is known about the replication fork arrest by thymine deprivation. In the case of UV irradiation, deficiency in any one of the RecFOR proteins renders the cell hypersensitive to UV [13,54,55], probably due to the cell’s inability to protect arrested replication forks [40]. By analogy, if inadequate protection of stalled replication forks should be mainly responsible for TLD, the recF and recO mutants are expected to be hypersensitive rather than resistant to TLD (Fig. 2). This appears to make the fork model less likely. However, it is conceivable that the fork failure may account for a fraction of TLD or, to be more specific, the residual TLD sensitivity of recF, recJ, recO, and recQ mutants [14,16,49]. Another corollary of this idea is that the above-mentioned recA conundrum may reflect the contribution of replication fork disruption. Thus, in recA mutants, replication forks stalled by thymine deficiency may be disrupted with increased frequencies, and the resulting increase in lethality would counter the repressive effect of RecA deficiency on the recombinationdependent lethal events involving the bulk DNA. This may account for the apparent indifference of the recA mutation to TLD [14].
7. Coda TLD is a ubiquitous phenomenon. It occurs in most, if not all, cells; even the monstrous bacterium Deinococcus radiodurans with an enormous capacity to repair DNA double-strand breaks is not free from it [56]. Our attempt to learn more about it culminated in the identification and characterization of the recQ gene and the gene product RecQ. Although the discovery of RecQ did not lead us directly to the final solution, it
certainly marked a significant step toward understanding TLD. It is hoped that a perfect answer to the puzzle could be obtained by developing the ideas discussed above.
Acknowledgements I thank K. Umezu and K. Nakayama for critical reading of the manuscript. K. Nakayama also kindly permitted me to cite his unpublished results. Comments from I. Grant and M. Inoue on the English of an earlier version of the manuscript are acknowledged with appreciation.
References [1] O. Maaløe, P.C. Hanawalt, Thymine deficiency and the normal DNA replication cycle. I, J. Mol. Biol. 3 (1961) 144–155. [2] H. Nakayama, A. Pratt, P. Hanawalt, Ultraviolet-induced repair synthesis in the duplicated and non-duplicated regions of the Escherichia coli chromosome, J. Mol. Biol. 70 (1972) 281–289. [3] P.C. Hanawalt, Involvement of synthesis of RNA in thymineless death, Nature 198 (1963) 286. [4] C. Pauling, P. Hanawalt, Nonconservative DNA replication in bacteria after thymine starvation, Proc. Natl. Acad. Sci. U.S.A. 54 (1965) 1728–1735. [5] D.W. Smith, P.C. Hanawalt, Macromolecular synthesis and thymineless death in Mycoplasma laidlawii B, J. Bacteriol. 96 (1968) 2066–2076. [6] S.S. Cohen, H.D. Barner, Studies on unbalanced growth in E. coli, Proc. Natl. Acad. Sci. U.S.A. 40 (1954) 885–893. [7] C.A. Coughlin, E.A. Adelberg, Bacterial mutation induced by thymine starvation, Nature 178 (1956) 531–532. [8] R. Weinberg, A.B. Latham, Apparent mutagenic effect of thymine deficiency for a thymine-requiring strain of Escherichia coli, J. Bacteriol. 72 (1956) 570–572. [9] D. Kanazir, The apparent mutagenicity of thymine deficiency, Biochim. Biophys. Acta 30 (1958) 20–23. [10] J. Gallant, T. Spottswood, Measurement of the stability of the repressor of alkaline phosphatase synthesis in E. coli, Proc. Natl. Acad. Sci. U.S.A. 52 (1964) 1591–1598. [11] D. Freifelder, Single-strand breaks in bacterial DNA associated with thymine starvation, J. Mol. Biol. 45 (1969) 1–7. [12] H. Nakayama, P. Hanawalt, Sedimentation analysis of deoxyribonucleic acid from thymine-starved Escherichia coli, J. Bacteriol. 121 (1975) 537–547. [13] Z.-I. Horii, A.J. Clark, Genetic analysis of the RecF pathway of genetic recombination in Escherichia coli K12: isolation and characterization of mutants, J. Mol. Biol. 80 (1973) 327–344. [14] H. Nakayama, K. Nakayama, R. Nakayama, Y. Nakayama, Recombination-deficient mutations and thymineless death in Escherichia coli K12: reciprocal effects of recBC and recF and
H. Nakayama / Mutation Research 577 (2005) 228–236
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26]
[27]
[28]
[29]
[30]
indifference of recA mutations, Can. J. Microbiol. 28 (1982) 425–430. H. Nakayama, K. Nakayama, R. Nakayama, N. Irino, Y. Nakayama, P.C. Hanawalt, Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway, Mol. Gen. Genet. 195 (1984) 474–480. K. Nakayama, N. Irino, H. Nakayama, The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants, Mol. Gen. Genet. 200 (1985) 266–271. N. Irino, K. Nakayama, H. Nakayama, The recQ gene of Escherichia coli K12: primary structure and evidence for SOS regulation, Mol. Gen. Genet. 205 (1986) 298–304. K. Umezu, K. Nakayama, H. Nakayama, Escherichia coli RecQ protein is a DNA helicase, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 5363–5367. A.E. Gorbalenya, E.V. Koonin, Helicases: amino acid sequence comparisons and structure-function relationships, Curr. Opin. Struct. Biol. 3 (1993) 419–429. D.A. Bernstein, J.L. Keck, Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family, Nucleic Acids Res. 31 (2003) 2778–2785. V. Morozov, A.R. Mushegian, E.V. Koonin, P. Bork, A putative nucleic acid-binding domain in Bloom’s and Werner’s syndrome helicases, Trends Biochem. Sci. 22 (1997) 417–418. D.A. Bernstein, M.C. Zittel, J.L. Keck, High-resolution structure of the E. coli RecQ helicase catalytic core, EMBO J. 22 (2003) 4910–4921. K. Umezu, H. Nakayama, RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein, J. Mol. Biol. 230 (1993) 1145–1150. F.G. Harmon, S.C. Kowalczykowski, Biochemical characterization of the DNA helicase activity of Escherichia coli RecQ helicase, J. Biol. Chem. 276 (2001) 232–243. R.R. Meyer, P.S. Laine, The single-stranded DNA-binding protein of Escherichia coli, Microbiol. Rev. 54 (1990) 342–380. F.G. Harmon, S.C. Kowalczykowski, RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination, Genes Dev. 12 (1998) 1134–1144. F.G. Harmon, R.J. DiGate, S.C. Kowalczykowski, RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination, Mol. Cell 3 (1999) 611–620. F.G. Harmon, J.P. Brockman, S.C. Kowalczykowski, RecQ helicase stimulates both DNA catenation and changes in DNA topology by topoisomerase III, J. Biol. Chem. 278 (2003) 42668–42678. A.S. Kamath-Loeb, L.A. Loeb, E. Johansson, P.M.J. Burgers, M. Fry, Interactions between the Werner syndrome helicase and DNA polymerase ␦ specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence, J. Biol. Chem. 276 (2001) 16439–16446. J.K. Karow, R.H. Newman, P.S. Freemont, I.D. Hickson, Oligomeric ring structure of the Bloom’s syndrome helicase, Current Biol. 9 (1999) 597–600.
235
[31] S. Huang, S. Beresten, B. Li, J. Oshima, N.A. Ellis, J. Campisi, Characterization of the human and mouse WRN 3 → 5 exonuclease, Nucleic Acids Res. 28 (2000) 2396–2405. [32] S. Cui, R. Klima, A. Ochem, D. Arosio, A. Falaschi, A. Vindigni, Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A, J. Biol. Chem. 278 (2003) 1424– 1432. [33] H.Q. Xu, E. Deprez, A.H. Zhang, P. Tauc, M.M. Ladjimi, J.C. Brochon, C. Auclair, X.G. Xi, The Escherichia coli RecQ helicase functions as a monomer, J. Biol. Chem. 278 (2003) 34925–34933. [34] K. Hanada, T. Ukita, Y. Kohno, K. Saito, J. Kato, H. Ikeda, RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3860–3865. [35] K. Shiraishi, K. Hanada, Y. Iwakura, H. Ikeda, Roles of RecJ, RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli, J. Bacteriol. 184 (2002) 4715–4721. [36] H. Ikeda, Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli, in: S. Ebashi (Ed.), Advances of Biophysics, vol. 38, Japan Scientific Societies Press, Elsevier, Tokyo, Limerick, 2004, pp. 3–20. [37] C. Luisi-DeLuca, S.T. Lovett, R.D. Kolodner, Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants, Genetics 122 (1989) 269–278. [38] J. Courcelle, P.C. Hanawalt, RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli, Mol. Gen. Genet. 262 (1999) 543–551. [39] J. Courcelle, J.R. Donaldson, K.-H. Chow, C.T. Courcelle, DNA damage-induced replication fork regression and processing in Escherichia coli, Science 299 (2003) 1064–1067. [40] K.-H. Chow, J. Courcelle, RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli, J. Biol. Chem. 279 (2004) 3492–3496. [41] T. Hishida, Y.-W. Han, T. Shibata, Y. Kubota, Y. Ishino, H. Iwasaki, H. Shinagawa, Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks, Genes Dev. 18 (2004) 1886–1897. [42] J. Courcelle, P.C. Hanawalt, RecA-dependent recovery of arrested DNA replication forks, Annu. Rev. Genet. 37 (2003) 611–646. [43] J. Courcelle, A.K. Ganesan, P.C. Hanawalt, Therefore, what are recombination proteins there for? BioEssays 23 (2001) 463–470. [44] S.T. Lovett, A.J. Clark, Genetic analysis of the recJ gene of Escherichia coli K-12, J. Bacteriol. 157 (1984) 190–196. [45] G.A. Cromie, C.B. Millar, K.H. Schmidt, D.R.F. Leach, Palindromes as substrates for multiple pathways of recombination in Escherichia coli, Genetics 154 (2000) 513–522. [46] S. Gangloff, J.P. McDonald, C. Bendixen, L. Arthur, R. Rothstein, The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase, Mol. Cell. Biol. 14 (1994) 8391–8398.
236
H. Nakayama / Mutation Research 577 (2005) 228–236
[47] Q. Zhu, P. Pongpech, R.J. DiGate, Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 9766–9771. [48] C.E. Deutch, C. Pauling, Thymineless mutagenesis in Escherichia coli, J. Bacteriol. 119 (1974) 861–867. [49] K. Nakayama, S. Shiota, H. Nakayama, Thymineless death in Escherichia coli mutants deficient in the RecF recombination pathway, Can. J. Microbiol. 34 (1988) 905–907. [50] K. Umezu, N.-W. Chi, R.D. Kolodner, Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 3875–3879. [51] K. Umezu, R.D. Kolodner, Protein interactions in genetic recombination in Escherichia coli, J. Biol. Chem. 269 (1994) 30005–30013. [52] K. Morimatsu, S.C. Kowalczykowski, RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand ex-
[53]
[54]
[55]
[56]
change: a universal step in recombinational repair, Mol. Cell 11 (2003) 1337–1347. K. Nakayama, K. Kusano, N. Irino, H. Nakayama, Thymine starvation-induced structural changes in Escherichia coli DNA. Detection by pulsed field gel electrophoresis and evidence for involvement of homologous recombination, J. Mol. Biol. 243 (1994) 611–620. R. Kolodner, R.A. Fishel, M. Howard, Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli, J. Bacteriol. 163 (1985) 1060–1066. A.A. Mahdi, R.G. Lloyd, Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair, Mol. Gen. Genet. 216 (1989) 503–510. J.G. Little, P.C. Hanawalt, Thymineless death and ultraviolet sensitivity in Micrococcus radiodurans, J. Bacteriol. 113 (1973) 233–240.