This enables it to anneal to the second end Fig. Following this route, COs are not possible. About 10 years ago, with meiotic recombination data from Saccharomyces cerevisiae , these two conflicting models were combined into one consolidated scheme, called the revised model Allers and Lichten, ; Hunter and Kleckner, Research in the past 20 years has shown that a number of different helicase families are involved in several steps of the HR reaction, and their loss can lead to critical problems in the cell.
In humans, mutations in those helicases have been implicated in development of cancer and debilitating hereditary diseases. Meiotic recombination is affected as well, when some of these helicases are missing. From a basic research point of view, it is important to decipher the similarities and differences in the functions of the proteins involved in genome stability and HR.
Since DNA repair and recombination is thought to have evolved very early, the research on those mechanisms on the plant side of eukaryotes can enable the comparison of these pathways in different kingdoms. Moreover, some DNA repair and recombination mutants that are embryo lethal in mammals are viable in plants, making meiotic studies possible W.
Li et al. There is also an applied aspect to this research in plants: taking control of the recombination reactions could enable researchers to improve plant biotechnology by using directed methods of genome modification. In the following sections, different classes of helicases will be discussed that are present in plants and play a role in somatic and meiotic recombination see Table 1 for homologues in humans and yeast, and Table 2 for published in vivo and in vitro data of the plant helicases discussed below.
The review will mainly focus on recent data found in plants. However, not all the enzymes discussed in this review have been characterized in detail in plants yet. As their presence in various plant species indicates their general importance, data obtained in other eukaryotes will also be referred to in order to describe their function. Where sequence comparisons, structural or functional data allow identification, homologues or analogues of the plant DNA helicases discussed in this review are given.
Given are all plant DNA helicases involved in somatic or meiotic homologous recombination that are mentioned in this review. Where applicable, the published phenotypes of the helicases are given, together with the relevant references.
See text for details. First described in a screen for thymineless death-resistant mutants in Escherichia coli Nakayama et al. Virtually all organisms tested so far possess at least one RecQ helicase gene, with increasing numbers of copies found in multicellular eukaryotes see Fig. The expression of E. Domain composition of selected RecQ family helicases.
The proteins are aligned at their helicase domains. Both are most probably needed for the recognition of and binding to unconventional DNA structures, e. Holliday junctions. For individual RecQ helicases, further domains have been described.
In animal homologues of RECQ5, a large C-terminal region seems to be conserved, although no function could yet be assigned. Finally, in plant RecQ helicases two domains can be identified bioinformatically. RECQsim also contains an insertion of mainly acidic residues in its helicase domain. In the yeast S. In humans, three of the five RecQ helicases are associated with severe hereditary diseases.
Mutations in the BLM gene result in Bloom syndrome BS , which is characterized by an elevated susceptibility to all types of cancers and several developmental defects Ellis et al. Here, the occurrence of some types of cancer is more prominent than in the normal population.
More interestingly, WS patients show a type of segmental progeria. From the middle of the second decade on, their ageing is accelerated, with typical age-related malignancies such as osteoporosis, diabetes mellitus, and cataracts occurring earlier in life Epstein et al.
There are intriguing results that open up the possibility that RECQL4 is important for the initiation of replication and could be a second replicative helicase beside the MCM complex in vertebrates Matsuno et al. Xu et al. This does not exclude a role for these proteins in DNA repair or recombination, though.
In plants, at least seven different RecQ helicases can be found Hartung et al. RECQsim is of intermediate length, but is unique compared with RecQ helicases in other kingdoms in that it has an insertion of mainly acidic amino acids in its helicase domain.
Additionally, in plants there is a small protein, WRNexo, with only an exonuclease domain that is most similar to the exonuclease domain found in the human WRN RecQ protein. Of these plant RecQ helicases and related proteins, still very little is known. Os RECQ1 has been shown to be required for transcription from loci that are able to form cruciform structures, possibly pointing to a role in RNA silencing H.
Chen et al. The expression of these three genes, and of rice RECQ2 , can be induced by genotoxic agents Saotome et al. Whether the latter two proteins indeed have a function in genome stability of chloroplast DNA awaits future mutant analysis. Testing more complex DNA substrates that resemble intermediates of DNA repair and recombination, the authors found differences in the activities of the two enzymes: RECQ2 resolves a partially mobile HJ structure in a way reminiscent of branch migration.
Additionally, both enzymes act on nicked HJs, but interestingly with different preferences concerning the outcome of the reaction. RECQ3, however, will unwind the lagging strand of a replication fork structure. Bypass of leading strand lesions by replication fork regression. When the replication fork encounters a lesion in the leading strand that cannot be overcome by the replicative DNA polymerase, one pathway to bypass the lesion and proceed with replication is post-replicative repair PRR; see text for details.
In the undamaged lagging strand, overshoot synthesis will elongate the nascent daughter strand past the position of the lesion in the leading strand. Because of the overshoot synthesis, the shorter of the strands can now be elongated.
After a reversal of the fork, the lesion on the leading strand has been bypassed without introducing a point mutation.
Alternatively, the chicken foot can also initiate a homologous recombination reaction in S-phase. However, despite these similarities, the two gene products seem to perform very different functions in the cell.
RECQ4A is comparable in many phenotypes with the yeast Sgs1 and the human BLM RecQ helicases; a knockout leads to hyper-recombination and increased sensitivity against a number of genotoxins. Expression of RECQ4A in a yeast sgs1 mutant suppresses its methylmethane sulphonate MMS sensitivity and brings its increased recombination rate down to wild-type levels Bagherieh-Najjar et al. A recq4B mutant, on the other hand, has so far not shown any defect in DNA repair, but has a reduced HR rate, a phenotype not known for any other RecQ helicase described so far Hartung et al.
Unfortunately, the sequence homologies are not strong enough for an unequivocal classification of all homologues. However, due to the fact that interactions are genetically conserved, at least for WRN and BLM the respective homologues in plants can be identified. Of all seven RecQ helicase proteins in A.
Hartung, personal communication. In yeast meiosis, both proteins are needed for the proper progression through meiotic recombination, and their loss leads to the accumulation of meiotic intermediates Jessop and Lichten, ; Oh et al.
This synthetic lethality is also conserved in Arabidopsis , where a recq4A mus81 double mutant will die shortly after germination Hartung et al. Dissolution was theoretically proposed as an alternative model for DSB repair by Thaler and Stahl , and it has received a form of renaissance in recent years.
However, instead of a dHJ resolution by an endonuclease, the dissolution reaction will produce a hemicatenane structure by using a DNA helicase to branch migrate the two HJs together Fig. A specialized type I topoisomerase can then open the hemicatenane to release the two dsDNA molecules Fig. The RecQ helicase is needed to transform two HJs into a hemicatenane that can then be resolved by the type I topoisomerase. Through protein interactions, RMI1 will stabilize the complex and indirectly enhance the dHJ dissolution reaction Chang et al.
This so-called RTR complex has been shown to be conserved in plants, too. All three genes share common DNA repair and recombination phenotypes consistent with the action of the RTR complexes in other organisms Hartung et al. For the first time in any eukaryote, an important role for members of the RTR complex in meiotic recombination could be shown in planta.
After apparent chromosome fragmentation in prophase I, fragmented DNA stays at the metaphase plate and is not moved to the poles in anaphase I, with DNA bridges connecting the chromosome fragments.
Both mutants never enter meiosis II and arrest at the end of meiosis I as dyads Chelysheva et al. While recq4A mutants do not display similar defects in meiotic recombination, a meiotic role for the helicase has been elucidated recently: RECQ4A localizes to telomeric foci during prophase I and is thought to dissolve telomere interactions between non-homologous chromosomes J. Higgins and F. Franklin, personal communication. Later, at the stage of the dHJ, for a second time during the recombination reaction a choice in direction to a NCO outcome becomes possible.
To describe meiotic HR fully, the revised model therefore has to be amended by the dissolution pathway, at least for A. The best understood eukaryotic anti-recombinase is the S. When it reaches the end of the D-loop, Srs2 starts to unwind the double strand.
This generates a single-stranded region on the template strand, where Srs2 can switch to. Stimulated by Rad51 bound to the invading strand, Srs2 will disrupt the heteroduplex strand by its helicase activity, thus enabling the now free extended end to anneal to the second end of the DSB, resulting in an NCO outcome via the SDSA pathway Dupaigne et al. While most of the work on Srs2 has been done in yeast, it seems to be conserved across most eukaryotes, with potential homologues also found in animals though missing in teleostei and mammals and in all plants.
An in vitro study recently showed that A. The same study showed, however, a new activity for At SRS2, which is capable of annealing of two single strands of DNA to a double strand. This can also be integrated into a recombination model: after the release of the single strand from the D-loop by SRS2, it can be annealed by the same protein to the second free end, which would be the following step in the SDSA pathway. However, no reports on the role of AtSRS2 in vivo have been published yet.
Apart from SRS2 homologues, other proteins have been described as anti-recombinases in eukaryotes. FBH1, which is found in many fungi but not S. Interestingly, both the helicase and the F-box domain are needed for this function Chiolo et al.
Not only are single mutant phenotypes similar between SRS2 and FBH1 , but also genetic and physical interactions with other genes or their products, respectively. Knocking out the single RecQ helicase of S. In Arabidopsis , a FBH1 homologue seems to be missing. There is only one protein with both an F-box and a helicase domain in the most recent release of the Arabidopsis genome, At3g G.
Recently, a further functional homologue of yeast Srs2 has been described in animals. Single nucleotide polymorphisms SNPs in RTEL1 have been associated with increased susceptibility to glioma and survival span in glioblastoma Shete et al. More basic studies on the role of RTEL1 in DNA repair and recombination have been done in Caenorhabditis elegans and in human cell culture, where it has been shown to share many phenotypes with SRS2 : a knockout or knockdown leads to defective DNA repair and hyper-recombination, and as is the case with FBH1 , lethal double mutants are also conserved Barber et al.
Recently, a role in meiotic recombination was identified for C. The study of the anti-recombinases SRS2 and RTEL1 and their interplay in plants promises interesting results, because yeast and mammals only possess homologues of either one or the other of these helicases. The human hereditary disease Fanconi anemia FA correlates with a broad spectrum of clinical phenotypes, including reduced numbers of all types of blood cells, leukaemia, and other forms of cancer and developmental defects.
Common cellular phenotypes are disturbances in the regulation of the cell cycle and apoptosis, spontaneous chromosome breaks and radial chromosomes, as well as sensitivity against interstrand cross-link-inducing genotoxins such as mitomycin C Neveling et al. Most of the FA proteins are grouped into the so-called core complex, which binds to stalled replication forks at sites of DNA interstrand cross-links. Interestingly, these are the FA genes for whose products a biochemical function could be shown.
Located at the N-terminus is a helicase domain for which ATPase activity, but no true unwinding activity, could be demonstrated. Interestingly, this translocase activity is able to branch migrate replication forks and HJs and to dissociate D-loops in vitro Meetei et al. Similar to the archaeal Hef protein, mammalian FANCM homologues possess an additional endonuclease domain at the C-terminus; mutations in key residues in this domain indicate that it is most probably inactive.
The baker's yeast FANCM homologue Mph1 is shorter than its mammalian counterpart, lacking the C-terminal region including the endonuclease domain. A FAAP24 homologue could not be identified so far. Mph1 also seems to be involved in the repair of DNA damage at stalled replication forks.
Image JSmol 1 A second protein, a helicase loader, assists the initiator and chaperones the replicative DNA helicase onto the single strand.
The structure of the helicase loader DnaC is remarkably similar to the structure of DnaA, forming a similar helix of subunits. You can use the "Compare Structures" tool to overlap these two structures, in PDB entries 3ecc and 3r8f.
References 4m4w: B. Liu, W. Steitz Structure of a helicase-helicase loader complex reveals insights into the mechanism of bacterial primosome assembly. Nature Communications 4: Itsathitphaisarn, R. Wing, W. Eliason, J. Steitz The hexameric helicase DnaB adopts a nonplanar conformation during translocation.
Cell , Soultanas Loading mechanisms of ring helicases at replication origins. Molecular Microbiology 84, Duderstadt, K. Later work 91 , 92 confirmed these observations, leading researchers to speculate that DOG-1 helicase might efficiently unwind G-quadruplex DNA structures that impede cellular DNA replication and pose a source of genomic instability.
The telomere loss characteristic of rtel mutant mice may be a direct consequence of defective DNA metabolism due to lack of specialized helicase action at the telomere end. In support of this, rtel-deficient mouse cells display elevated spontaneous fragile telomeres Purified recombinant human RTEL can actively disrupt three-stranded D-loop recombination intermediates, which may be relevant to its putative anti-recombinase role Determination if RTEL associates with components of the shelterin complex and the functional consequences of the interaction should be revealing for finding clues to the potential involvement of the helicase in the prevention of a human telomere disease.
A number of telomere diseases are known Of particular interest is dyskeratosis congenita DC , which is defective in the DKC1 gene that encodes dyskerin 99 , an RNA-binding protein that is a component of the telomerase complex The Dillingham lab first reported a novel class of proteins with a nuclease domain that contains a conserved Fe—S cluster.
Four conserved cysteines flank the conserved RecB family nuclease domain. Given the importance of nucleases in DNA processing events associated with DNA repair and replication, the discovery of helicase—nuclease enzymes with conserved Fe—S clusters has sparked great interest in their structure and function.
Positions of the four conserved cysteine residues in the nuclease domain green are shown for human Dna2 and Bacillus subtilis AddB The helicase domain is shown in purple. For Dna2, the nuclease and helicase domains reside in the same polypeptide. AddA contains a second nuclease domain as well as a helicase domain. Like E. The dual nuclease reaction mechanism is regulated by a consensus DNA sequence element known as cross-over hotspot instigator Chi.
The AddB Fe—S domain contains four conserved cysteine residues with the first cysteine residing N-terminal to the nuclease domain and the other three cysteines residing on the C-terminal side. Characterization of the Fe—S mutants revealed that the AddAB protein complex contains a subane [4Fe—4S] cluster which serves as a molecular staple to stabilize the AddB nuclease domain A critically important advance for understanding the structural and functional significance of the iron staple domain will be to solve the structure of the AddAB protein—DNA complex.
This should yield new insight to the architecture and function of AddAB and perhaps other specialized DNA processing enzymes, such as eukaryotic Dna2 that also possesses a conserved Fe—S staple domain , in DSB strand resection step and other aspects of DNA metabolism. Although mutations in the Fe—S cluster did not affect AddAB ATPase or translocase activity, the presence of an Fe—S domain affected the protein complex's ability to bind broken DNA, a result that is likely to be relevant to the function of the conserved iron staple nuclease domain in other DNA repair proteins, including the SF1 Dna2 helicase—nuclease Figure 5 , a eukaryotic protein implicated in DSB end resection, Okazaki fragment processing and telomere stabilization , Given that Dna2 is essential for cellular DNA replication, it seems likely that mutations in the Fe—S domain of Dna2 will be poorly tolerated.
Although biochemical studies with purified proteins and genetic analysis in yeast suggest that a Dna2-independent EXO1-dependent pathway of DSB end resection exists , , the endonucleoytic function provided by the Dna2 pathway may be important for strand resection of DNA sequences in a BLM helicase-dependent manner It will be incumbent to extract information from biochemical and structural studies of AddAB and apply this insight to the related iron staple nuclease Dna2.
Recent studies have provided evidence that in addition to its nuclear functions, Dna2 can be found in mitochondria and is important for flap processing during BER and the fidelity of DNA replication in mitochondria , Understanding how Dna2 balances its duties between mitochondria and the nucleus is a high priority. Presumably, the Fe—S domain of Dna2 is required for enzymatic function and its cellular roles; however, no biochemical or genetic studies have directly tested this.
A defect in the synthesis of Fe—S clusters is responsible for mitochondrial dysfunction, leading to nuclear genomic instability Further studies are required to ascertain the importance of the Fe—S staple domain in Dna2 for its nuclear and mitochondrial functions, and the influence of iron homeostasis in this respect.
It has been speculated that DNA repair proteins with Fe—S clusters may use their redox properties to scan the genome for DNA damage by sensing DNA charge transport that is mediated by base pair stacking For example, it was proposed that the bacterial redox-sensitive transcription regulator SoxR becomes activated by DNA-mediated charge transport Fe—S cluster BER enzymes e.
An intriguing aspect of both SoxR and the DNA repair glycosylases is that their redox activities are altered upon DNA binding 84 , 85 , suggesting a possible mechanism for assaying DNA-mediated signaling. This may be relevant to Fe—S cluster helicases and helicase—nucleases, particularly those that play a role in DNA damage recognition or verification.
The signal intensity corresponded to the equivalent of a one-electron redox couple of the 4Fe—4S cluster. An important advance was made by the demonstration that SaXPD displayed ATP-dependent electrochemistry, consistent with the idea that the electrochemical signal is coupled to mechanical movement of the helicase as it translocates on DNA. In addition to its structural role, the Fe—S cluster may be important for sensing unusual DNA structures whether it is a covalent base adduct or sequence element with an alternate conformation.
In terms of base damage, FANCJ is capable of sensing an oxidative base damage thymine glycol in either the translocating or non-translocating strands of duplex DNA Understanding how the redox activity of the Fe—S cluster plays a role in DNA recognition is an important question. While a specific mechanism has not yet been elucidated, a theoretical study of DNA damage recognition through electron transfer mediated by the 4Fe—4S complex of the DNA glycolsylase MutY that builds upon the studies of the Barton lab suggested a setting in which charge transfer stabilizes a specific conformation of the protein that places it in the recognition mode preferentially over the non-specific binding conformation, providing a means for the DNA repair protein to localize the damaged site in an efficient manner This model proposes that in order for electron transfer mediated by the Fe—S redox activity to be relevant to the process of scanning DNA for sites of damage, the rate of electron transport from donor to acceptor should be greater than the rate of protein diffusion along DNA.
Such electron tunneling between DNA repair proteins may be mediated by efficient electron transport through the DNA; however, this theory remains to be proven. Redox signaling by Fe—S cluster helicases may also play a role in the catalytic reaction mechanism.
A hypothesis which remains to be tested is that Fe—S cluster helicase molecules bound to DNA may communicate with each other by changes in redox activity that are mediated through DNA charge transport Figure 6. The groundwork for this hypothesis was provided by the Raney lab which provided evidence that the Dda helicase monomer, which incidentally lacks an Fe—S cluster, functionally cooperates with other DNA-bound monomers They were able to show that increasing the length of the single-stranded DNA overhang enhanced unwinding of the adjacent duplex by Dda.
Their results suggested a model in which multiple Dda molecules bound to the same substrate displayed greater processivity for DNA unwinding.
Similarly, multiple NS3 helicase molecules bound to the single-stranded DNA loading region of a partial duplex substrate are required for optimal unwinding More recently, biochemical studies have provided evidence for functional cooperativity between E. Although Dda, NS3 and RecQ helicases lack the conserved Fe—S cluster, these proteins may utilize other motifs to communicate between helicase molecules by DNA-mediated redox signaling or another mechanism.
Nonetheless, it will be of interest to determine if Fe—S cluster helicase molecules functionally cooperate during DNA unwinding and if the redox function plays a role. Models are adapted by analogy from ones proposed for bacteriophage T4 Dda helicase , In addition to DNA unwinding, multiple helicase molecules loaded on the same single-stranded DNA molecule may cooperate to facilitate protein displacement , a function that may be relevant to the role of certain helicases to facilitate DNA replication or transcription, as mentioned earlier.
It has been proposed that the presence of multiple motors may serve to prevent backward displacement on the single-stranded DNA, resulting in an elevated force production to displace protein from the DNA molecule. It is conceivable that Fe—S cluster helicases have emerged as a group of proteins that utilize their ability to alter redox potential in order to communicate between DNA-bound helicase molecules during the task of protein blockade clearance Figure 6.
For example, the ability of certain Fe—S helicases e. It will be of interest to determine if any of the Fe—S cluster helicases behave in a cooperative manner, and if the redox activity of the Fe—S cluster plays a role in this capacity.
Whether or not the redox activity of the FANCJ Fe—S cluster plays a role in the unwinding mechanism remains to be experimentally tested. Single turnover kinetic analyses of DNA helicases such as FANCJ and selected site-directed Fe—S cluster mutants should provide insight to the possibility that the helicase monomers display functional cooperativity in order to efficiently perform their unwinding or protein displacement functions.
Nonetheless, it is provocative to consider that the Fe—S cluster may constitute a specialized structural element that helps to dictate the directionality of DNA translocation for the Fe—S domain helicases. The crystal structure of TaXPD bound to single-stranded DNA Figure 3 45 suggests that translocation directionality is dictated by conformational changes within the motor domain that determine ATP-driven enzyme translocation in a directional manner.
There is very little information yet concerning the functional importance of the Fe—S cluster in helicase—nuclease enzymes. One hypothesis is that the conserved Fe—S staple domain in AddAB may regulate the nuclease reaction mechanism by sensing recombination hotspots via alterations in its redox activity at consensus DNA sequence elements. It is anticipated that the endonuclease activity catalyzed by Dna2 necessary for Okazaki fragment processing is tightly regulated and may also involve redox signaling mediated by the Fe—S cluster domain.
Given the likelihood that DNA charge transport plays an important role in the movement of Fe—S cluster proteins during DNA damage surveillance, it seems probable that Fe—S cluster helicases would exploit the redox active domain for its catalytic functions in ATP-dependent DNA duplex unwinding or protein displacement.
This is clearly an important area of investigation as it will lead to new insights to the mechanism of action of Fe—S cluster helicases, which are required for a normal level of DNA repair and maintenance of genomic stability. We thank these scientists and Dr Malcolm White for helpful discussions. National Center for Biotechnology Information , U. Journal List Nucleic Acids Res v. Nucleic Acids Res.
Published online Jan Robert M. Brosh, Jr. Author information Article notes Copyright and License information Disclaimer. Correspondence may also be addressed to Yuliang Wu. Copyright Published by Oxford University Press This article has been cited by other articles in PMC. Abstract Conserved Iron—Sulfur Fe—S clusters are found in a growing family of metalloproteins that are implicated in prokaryotic and eukaryotic DNA replication and repair.
Open in a separate window. Figure 1. Figure 2. Figure 3. DinG Sequence analysis of the E. Figure 4. Figure 5. Dna2 Although mutations in the Fe—S cluster did not affect AddAB ATPase or translocase activity, the presence of an Fe—S domain affected the protein complex's ability to bind broken DNA, a result that is likely to be relevant to the function of the conserved iron staple nuclease domain in other DNA repair proteins, including the SF1 Dna2 helicase—nuclease Figure 5 , a eukaryotic protein implicated in DSB end resection, Okazaki fragment processing and telomere stabilization , Figure 6.
Conflict of interest statement. None declared. Py B, Barras F. Building Fe-S proteins: bacterial strategies. Lill R. Function and biogenesis of iron-sulphur proteins. Ye H, Rouault TA. Erythropoiesis and iron sulfur cluster biogenesis.
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