COMMENTARY
COMMENTARY
Fine tuning of a DNA fork by the RecQ helicase Alicia K. Byrd1 and Kevin D. Raney1 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
Helicases are enzymes that couple the hydrolysis of ATP to the unwinding of duplex nucleic acids (NAs), thus providing the single-stranded NA (ssNA) intermediates necessary for nucleic acid processing and maintenance such as replication, recombination, repair, and transcription (1–3). All helicases possess a core helicase domain containing RecA-like motifs that are responsible for NA binding and ATP hydrolysis. Variation among individual helicases and helicase families within both the helicase core and in accessory domains allows this single class of enzymes to perform a myriad of different functions within the cell in different manners on various substrate types, resulting in helicases specific for every process involving NA in the cell. In PNAS, Rad et al. (4) provide a close-up view of a member of the RecQ family of enzymes that can modulate its activity to “fine tune” unwinding at a DNA fork. When DNA needs to be unwound, a helicase is typically involved. Unwinding produces a DNA fork structure, but the number of base pairs melted and the rate of melting are modulated to fit the needed task. For example, DNA replication requires a highly processive helicase activity, whereas some forms of DNA repair might require only a few base pairs to be melted. DNA recombination can involve unwinding of a
few base pairs, or thousands of base pairs. Some helicases, such as the hexameric replicative helicases, are highly processive, unwinding kilobases of DNA at a time (5). However, many of the nonhexameric helicases in superfamilies 1 and 2 are typically nonprocessive, often unwinding only a few base pairs in a single binding event (6). Some notable exceptions to this are the superfamily 1 helicases RecBCD, which unwinds 30 kb on average before dissociation (7), and TraI, which can unwind hundreds of base pairs as a monomer (8). The RecQ family of helicases is involved in recombination, repair, and replication in both prokaryotes and eukaryotes (9). Humans have five RecQ helicases: RECQL1, BLM, WRN, RECQL4, and RECQL5. Defects in three of these, BLM, WRN, and RECQL4, are associated with diseases characterized by premature aging and cancer (10). The RecQ family has many biological roles. Escherichia coli RecQ is an unusual helicase in that it can unwind closed circular DNA (11), whereas most helicases require an ssDNA tail or sometimes can initiate unwinding at a dsDNA end. RecQ can both initiate homologous recombination and disrupt joint molecules by branch migration (12). Additionally, in conjunction with topoisomerase III, RecQ unwinds and promotes
catenation of closed circular dsDNA (13) and resolves converging replication forks (14). Visualization of DNA unwinding at the single-molecule level has been accomplished using a number of systems in which total internal reflection fluorescence microscopy is applied. In single-molecule FRET (smFRET), the DNA substrate is typically visualized by labeling oligonucleotides with fluorescent probes such that appropriate FRET measurements can be made, leading to mechanistic conclusions. More recently, the proteins involved in the DNA unwinding process have been labeled with fluorescent probes to directly visualize events associated with protein binding and/or movement along the DNA. Such methods have provided stunning images and movies that greatly enable discernment of helicase mechanism(s). Rad et al. (4) developed an elegant system that allowed them to directly visualize the appearance of ssDNA during the unwinding process. They took advantage of the fact that the ssDNA binding protein (SSB) will associate with the newly created ssDNA after unwinding at the fork by RecQ. By labeling the SSB with fluorescent probes, unwinding forks appeared as fluorescent spots of varying intensity due to the accumulation of fluorescently labeled SSB. This approach allowed an important question to be addressed: How can the appropriate kinetic outcome for DNA unwinding be matched with a specific biological function? In the case of RecQ, the answer seems to depend on cooperative protein interactions. Quantitative analysis of the protein concentration dependence for rates of unwinding individual DNA forks revealed that varying numbers of RecQ molecules participate in different unwinding events. The results provide an avenue for interpreting how a helicase can fulfill diverse roles by assembly, with different kinetic outcomes dependent on the number of proteins bound at the DNA fork (Fig. 1). Author contributions: A.K.B. and K.D.R. wrote the paper. The authors declare no conflict of interest.
Fig. 1. Tuning a DNA fork. RecQ “tunes” the DNA fork differently depending on the number of RecQ monomers that assemble at the fork. A single monomer can slowly unwind the fork, but an assembly of at least four monomers results in rapid and processive fork propagation.
www.pnas.org/cgi/doi/10.1073/pnas.1520119112
See companion article 10.1073/pnas.1518028112. 1
To whom correspondence may be addressed. Email: akbyrd@ uams.edu or [email protected].
PNAS Early Edition | 1 of 2
Importantly, the simultaneous observation of multiple, separate DNA forks, with some moving at different speeds in the same experiment, provides convincing evidence for the variable “tuning” of a DNA fork by RecQ helicase. These different speeds are the result of assembly of varying numbers of RecQ helicases loosely associating at the DNA fork, sort of “DNA unwinding by committee.” RecQ is able to unwind dsDNA as a monomer (15, 16), but on substrates with an ssDNA tail it also exhibits functional cooperativity (17) and nearly stoichiometric amounts of RecQ are required for maximal unwinding (11). Evidence for functional cooperativity from ensemble experiments has been presented for bacteriophage T4 Dda (18, 19) and hepatitis C virus NS3h (20) in addition to RecQ. RecQ can initiate DNA melting at internal sites, leading to formation of one (unidirectional) or two (bidirectional) forks proceeding from the initiation site. A RecQ dimer seems to be needed to start the process on duplex DNA, but additional monomers can bind to the fork, with four or more monomers resulting in maximal rates of unwinding. Thus, the number of molecules at the fork, working together, can alter the rate and processivity of unwinding (Fig. 1). Major challenges in moving forward include establishing that
2 of 2 | www.pnas.org/cgi/doi/10.1073/pnas.1520119112
these mechanisms involving assemblies of helicases are functionally significant in cells and ultimately to relate different assembly states to specific biological processes. This paper from Rad et al. (4) and other recent papers (21, 22) illustrate how direct visualization of proteins on DNA can provide insight that ensemble approaches cannot reveal. Visualizing the DNA substrate
ACKNOWLEDGMENTS. This work was supported by NIH Grant R01 GM098922 (to K.D.R.).
1 Singleton MR, Dillingham MS, Wigley DB (2007) Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 76:23–50. 2 Lohman TM, Tomko EJ, Wu CG (2008) Non-hexameric DNA helicases and translocases: Mechanisms and regulation. Nat Rev Mol Cell Biol 9(5):391–401. 3 Pyle AM (2008) Translocation and unwinding mechanisms of RNA and DNA helicases. Annu Rev Biophys 37:317–336. 4 Rad B, Forget AL, Baskin RJ, Kowalczykowski SC (2015) Singlemolecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding. Proc Natl Acad Sci USA, 10.1073/pnas.1518028112. 5 Patel SS, Pandey M, Nandakumar D (2011) Dynamic coupling between the motors of DNA replication: Hexameric helicase, DNA polymerase, and primase. Curr Opin Chem Biol 15(5):595–605. 6 Raney KD, Byrd AK, Aarattuthodiyil S (2013) Structure and mechanisms of SF1 DNA helicases. Adv Exp Med Biol 767:17–46. 7 Roman LJ, Eggleston AK, Kowalczykowski SC (1992) Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme. J Biol Chem 267(6):4207–4214. 8 Lahue EE, Matson SW (1988) Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction. J Biol Chem 263(7):3208–3215. 9 Croteau DL, Popuri V, Opresko PL, Bohr VA (2014) Human RecQ helicases in DNA repair, recombination, and replication. Annu Rev Biochem 83:519–552. 10 Brosh RM, Jr, Bohr VA (2007) Human premature aging, DNA repair and RecQ helicases. Nucleic Acids Res 35(22):7527–7544. 11 Harmon FG, Kowalczykowski SC (2001) Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase. J Biol Chem 276(1):232–243.
12 Harmon FG, Kowalczykowski SC (1998) RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination. Genes Dev 12(8):1134–1144. 13 Harmon FG, DiGate RJ, Kowalczykowski SC (1999) RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: A conserved mechanism for control of DNA recombination. Mol Cell 3(5):611–620. 14 Suski C, Marians KJ (2008) Resolution of converging replication forks by RecQ and topoisomerase III. Mol Cell 30(6):779–789. 15 Xu HQ, et al. (2003) The Escherichia coli RecQ helicase functions as a monomer. J Biol Chem 278(37):34925–34933. 16 Zhang XD, et al. (2006) Escherichia coli RecQ is a rapid, efficient, and monomeric helicase. J Biol Chem 281(18):12655–12663. 17 Li N, et al. (2010) Multiple Escherichia coli RecQ helicase monomers cooperate to unwind long DNA substrates: A fluorescence crosscorrelation spectroscopy study. J Biol Chem 285(10):6922–6936. 18 Byrd AK, Raney KD (2004) Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA. Nat Struct Mol Biol 11(6):531–538. 19 Byrd AK, Raney KD (2006) Displacement of a DNA binding protein by Dda helicase. Nucleic Acids Res 34(10):3020–3029. 20 Levin MK, Wang YH, Patel SS (2004) The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. J Biol Chem 279(25):26005–26012. 21 Comstock MJ, et al. (2015) Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme. Science 348(6232):352–354. 22 Lee KS, Balci H, Jia H, Lohman TM, Ha T (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4:1878.
and the proteins at the DNA fork takes the old adage “seeing is believing” to the molecular level. At that level, we now can say that some helicases have the ability to fine tune a DNA fork in vitro possibly to suit specific biological functions.
Byrd and Raney
COMMENTARY
Fine tuning of a DNA fork by the RecQ helicase Alicia K. Byrd1 and Kevin D. Raney1 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
Helicases are enzymes that couple the hydrolysis of ATP to the unwinding of duplex nucleic acids (NAs), thus providing the single-stranded NA (ssNA) intermediates necessary for nucleic acid processing and maintenance such as replication, recombination, repair, and transcription (1–3). All helicases possess a core helicase domain containing RecA-like motifs that are responsible for NA binding and ATP hydrolysis. Variation among individual helicases and helicase families within both the helicase core and in accessory domains allows this single class of enzymes to perform a myriad of different functions within the cell in different manners on various substrate types, resulting in helicases specific for every process involving NA in the cell. In PNAS, Rad et al. (4) provide a close-up view of a member of the RecQ family of enzymes that can modulate its activity to “fine tune” unwinding at a DNA fork. When DNA needs to be unwound, a helicase is typically involved. Unwinding produces a DNA fork structure, but the number of base pairs melted and the rate of melting are modulated to fit the needed task. For example, DNA replication requires a highly processive helicase activity, whereas some forms of DNA repair might require only a few base pairs to be melted. DNA recombination can involve unwinding of a
few base pairs, or thousands of base pairs. Some helicases, such as the hexameric replicative helicases, are highly processive, unwinding kilobases of DNA at a time (5). However, many of the nonhexameric helicases in superfamilies 1 and 2 are typically nonprocessive, often unwinding only a few base pairs in a single binding event (6). Some notable exceptions to this are the superfamily 1 helicases RecBCD, which unwinds 30 kb on average before dissociation (7), and TraI, which can unwind hundreds of base pairs as a monomer (8). The RecQ family of helicases is involved in recombination, repair, and replication in both prokaryotes and eukaryotes (9). Humans have five RecQ helicases: RECQL1, BLM, WRN, RECQL4, and RECQL5. Defects in three of these, BLM, WRN, and RECQL4, are associated with diseases characterized by premature aging and cancer (10). The RecQ family has many biological roles. Escherichia coli RecQ is an unusual helicase in that it can unwind closed circular DNA (11), whereas most helicases require an ssDNA tail or sometimes can initiate unwinding at a dsDNA end. RecQ can both initiate homologous recombination and disrupt joint molecules by branch migration (12). Additionally, in conjunction with topoisomerase III, RecQ unwinds and promotes
catenation of closed circular dsDNA (13) and resolves converging replication forks (14). Visualization of DNA unwinding at the single-molecule level has been accomplished using a number of systems in which total internal reflection fluorescence microscopy is applied. In single-molecule FRET (smFRET), the DNA substrate is typically visualized by labeling oligonucleotides with fluorescent probes such that appropriate FRET measurements can be made, leading to mechanistic conclusions. More recently, the proteins involved in the DNA unwinding process have been labeled with fluorescent probes to directly visualize events associated with protein binding and/or movement along the DNA. Such methods have provided stunning images and movies that greatly enable discernment of helicase mechanism(s). Rad et al. (4) developed an elegant system that allowed them to directly visualize the appearance of ssDNA during the unwinding process. They took advantage of the fact that the ssDNA binding protein (SSB) will associate with the newly created ssDNA after unwinding at the fork by RecQ. By labeling the SSB with fluorescent probes, unwinding forks appeared as fluorescent spots of varying intensity due to the accumulation of fluorescently labeled SSB. This approach allowed an important question to be addressed: How can the appropriate kinetic outcome for DNA unwinding be matched with a specific biological function? In the case of RecQ, the answer seems to depend on cooperative protein interactions. Quantitative analysis of the protein concentration dependence for rates of unwinding individual DNA forks revealed that varying numbers of RecQ molecules participate in different unwinding events. The results provide an avenue for interpreting how a helicase can fulfill diverse roles by assembly, with different kinetic outcomes dependent on the number of proteins bound at the DNA fork (Fig. 1). Author contributions: A.K.B. and K.D.R. wrote the paper. The authors declare no conflict of interest.
Fig. 1. Tuning a DNA fork. RecQ “tunes” the DNA fork differently depending on the number of RecQ monomers that assemble at the fork. A single monomer can slowly unwind the fork, but an assembly of at least four monomers results in rapid and processive fork propagation.
www.pnas.org/cgi/doi/10.1073/pnas.1520119112
See companion article 10.1073/pnas.1518028112. 1
To whom correspondence may be addressed. Email: akbyrd@ uams.edu or [email protected].
PNAS Early Edition | 1 of 2
Importantly, the simultaneous observation of multiple, separate DNA forks, with some moving at different speeds in the same experiment, provides convincing evidence for the variable “tuning” of a DNA fork by RecQ helicase. These different speeds are the result of assembly of varying numbers of RecQ helicases loosely associating at the DNA fork, sort of “DNA unwinding by committee.” RecQ is able to unwind dsDNA as a monomer (15, 16), but on substrates with an ssDNA tail it also exhibits functional cooperativity (17) and nearly stoichiometric amounts of RecQ are required for maximal unwinding (11). Evidence for functional cooperativity from ensemble experiments has been presented for bacteriophage T4 Dda (18, 19) and hepatitis C virus NS3h (20) in addition to RecQ. RecQ can initiate DNA melting at internal sites, leading to formation of one (unidirectional) or two (bidirectional) forks proceeding from the initiation site. A RecQ dimer seems to be needed to start the process on duplex DNA, but additional monomers can bind to the fork, with four or more monomers resulting in maximal rates of unwinding. Thus, the number of molecules at the fork, working together, can alter the rate and processivity of unwinding (Fig. 1). Major challenges in moving forward include establishing that
2 of 2 | www.pnas.org/cgi/doi/10.1073/pnas.1520119112
these mechanisms involving assemblies of helicases are functionally significant in cells and ultimately to relate different assembly states to specific biological processes. This paper from Rad et al. (4) and other recent papers (21, 22) illustrate how direct visualization of proteins on DNA can provide insight that ensemble approaches cannot reveal. Visualizing the DNA substrate
ACKNOWLEDGMENTS. This work was supported by NIH Grant R01 GM098922 (to K.D.R.).
1 Singleton MR, Dillingham MS, Wigley DB (2007) Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 76:23–50. 2 Lohman TM, Tomko EJ, Wu CG (2008) Non-hexameric DNA helicases and translocases: Mechanisms and regulation. Nat Rev Mol Cell Biol 9(5):391–401. 3 Pyle AM (2008) Translocation and unwinding mechanisms of RNA and DNA helicases. Annu Rev Biophys 37:317–336. 4 Rad B, Forget AL, Baskin RJ, Kowalczykowski SC (2015) Singlemolecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding. Proc Natl Acad Sci USA, 10.1073/pnas.1518028112. 5 Patel SS, Pandey M, Nandakumar D (2011) Dynamic coupling between the motors of DNA replication: Hexameric helicase, DNA polymerase, and primase. Curr Opin Chem Biol 15(5):595–605. 6 Raney KD, Byrd AK, Aarattuthodiyil S (2013) Structure and mechanisms of SF1 DNA helicases. Adv Exp Med Biol 767:17–46. 7 Roman LJ, Eggleston AK, Kowalczykowski SC (1992) Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme. J Biol Chem 267(6):4207–4214. 8 Lahue EE, Matson SW (1988) Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction. J Biol Chem 263(7):3208–3215. 9 Croteau DL, Popuri V, Opresko PL, Bohr VA (2014) Human RecQ helicases in DNA repair, recombination, and replication. Annu Rev Biochem 83:519–552. 10 Brosh RM, Jr, Bohr VA (2007) Human premature aging, DNA repair and RecQ helicases. Nucleic Acids Res 35(22):7527–7544. 11 Harmon FG, Kowalczykowski SC (2001) Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase. J Biol Chem 276(1):232–243.
12 Harmon FG, Kowalczykowski SC (1998) RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination. Genes Dev 12(8):1134–1144. 13 Harmon FG, DiGate RJ, Kowalczykowski SC (1999) RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: A conserved mechanism for control of DNA recombination. Mol Cell 3(5):611–620. 14 Suski C, Marians KJ (2008) Resolution of converging replication forks by RecQ and topoisomerase III. Mol Cell 30(6):779–789. 15 Xu HQ, et al. (2003) The Escherichia coli RecQ helicase functions as a monomer. J Biol Chem 278(37):34925–34933. 16 Zhang XD, et al. (2006) Escherichia coli RecQ is a rapid, efficient, and monomeric helicase. J Biol Chem 281(18):12655–12663. 17 Li N, et al. (2010) Multiple Escherichia coli RecQ helicase monomers cooperate to unwind long DNA substrates: A fluorescence crosscorrelation spectroscopy study. J Biol Chem 285(10):6922–6936. 18 Byrd AK, Raney KD (2004) Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA. Nat Struct Mol Biol 11(6):531–538. 19 Byrd AK, Raney KD (2006) Displacement of a DNA binding protein by Dda helicase. Nucleic Acids Res 34(10):3020–3029. 20 Levin MK, Wang YH, Patel SS (2004) The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. J Biol Chem 279(25):26005–26012. 21 Comstock MJ, et al. (2015) Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme. Science 348(6232):352–354. 22 Lee KS, Balci H, Jia H, Lohman TM, Ha T (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4:1878.
and the proteins at the DNA fork takes the old adage “seeing is believing” to the molecular level. At that level, we now can say that some helicases have the ability to fine tune a DNA fork in vitro possibly to suit specific biological functions.
Byrd and Raney
