Architecture of the Replication Complex and DNA Loops at the Fork Generated by the Bacteriophage T4 Proteins

Abstract

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures (“bobbins”). These bobbins constitute a major portion of the mass of the full replication complex. Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures (“bobbins”). These bobbins constitute a major portion of the mass of the full replication complex. DNA replication is accomplished by a highly organized DNA-protein ensemble, operating as a molecular machine that replicates both strands in a highly coordinated manner. How the large number of protein species at a DNA replication fork, perhaps as many as 20 in Escherichia coli, and several kilobases of DNA strands, are arranged remains poorly understood. To accommodate the requirement that the two strands must be copied in the same enzymatic (5′ to 3′) but opposite physical direction, Alberts and co-workers (1Sinha N.K. Morris C.F. Alberts B.M. J. Biol. Chem. 1980; 255: 4290-4293Abstract Full Text PDF PubMed Google Scholar, 2Alberts B.M. Barry J. Bedinger P. Formosa T. Jongeneel C.V. Kreuzer K.N. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 655-668Crossref PubMed Google Scholar, 3Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google Scholar) proposed that the polymerase synthesizing the lagging strand remains in contact with the leading strand polymerase at the fork. This would fold the lagging strand into a loop and couple leading and lagging strand synthesis. The loop would grow as the Okazaki fragment lengthens, then collapse as the replication machinery moves from the mature Okazaki fragment to the next to be completed. Because of the visual resemblance to a trombone slide, this model has been termed the “trombone model.” Evidence for the coupling of leading and lagging strand synthesis and a lagging strand loop has come from physical and biochemical studies. The presence of two DNA polymerase III cores within one holoenzyme particle has been demonstrated in the E. coli replication system (4Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 5Wu C.A. Zechner E.L. Hughes Jr., A.J. Franden M.A. McHenry C.S. Marians K.J. J. Biol. Chem. 1992; 267: 4064-4073Abstract Full Text PDF PubMed Google Scholar, 6Maki H. Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6570-6578Abstract Full Text PDF PubMed Google Scholar, 7Glover B.P. McHenry C.S. Cell. 2001; 105: 925-934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and both polymerases within the complex can function simultaneously (4Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Early evidence for the coordination of leading and lagging strand synthesis obtained in E. coli (5Wu C.A. Zechner E.L. Hughes Jr., A.J. Franden M.A. McHenry C.S. Marians K.J. J. Biol. Chem. 1992; 267: 4064-4073Abstract Full Text PDF PubMed Google Scholar, 8Tougu K. Marians K.J. J. Biol. Chem. 1996; 271: 21398-21405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Kim S. Dallmann H.G. McHenry C.S. Marians K.J. Cell. 1996; 84: 643-650Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), T4 (2Alberts B.M. Barry J. Bedinger P. Formosa T. Jongeneel C.V. Kreuzer K.N. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 655-668Crossref PubMed Google Scholar, 10Schrock R.D. Alberts B. J. Biol. Chem. 1996; 271: 16678-16682Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 11Richardson R.W. Ellis R.L. Nossal N.G. UCLA Symp. Mol. Cell. Biol. 1990; 127: 247-259Google Scholar), and T7 (12Debyser Z. S. Cell. Full Text PDF PubMed Scopus Google Scholar) that the of Okazaki fragments not of the polymerase the evidence has come from with the purified T7 proteins and a circular in which into the leading and lagging strands be of a strand J. J. Mol. Biol. PubMed Scopus Google Scholar, J. T. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The of a of leading strand and synthesis of the lagging this that synthesis in a circle a coupling of synthesis of both with the T4 replication proteins Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the purified T7 replication proteins the DNA-protein complexes engaged in circle replication from circular templates containing preformed forks been examined by electron electron electron M13 of kb K. Z. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the J. J. Mol. Biol. PubMed Scopus Google Scholar, J. T. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) these a DNA loop at the fork with a replication complex. using these molecules that the replication at the from and are the of the lagging strand folding back both templates the loop size and with the duplex portion of a single Okazaki fragment as by the of the DNA strands and proteins at a replication fork. the with the T7 system to T7 typical of complex as T4 and E. In the T7 the single-stranded DNA segments at the fork not extended as in the but rather in compact that termed This the of the T7 gene protein as to the of the T4 gene 32 E. coli proteins to a of DNA replication T4 a with which to the of the with is by 10 different proteins a and system typical of E. coli and complex of the 10 proteins has been purified and in N.G. of and 2001; PubMed Scopus Google Scholar). both strands is by T4 DNA a with to The protein a as a to the of protein as a that to the Nossal N.G. Cell. Full Text PDF PubMed Scopus Google Scholar, Alberts B.M. J. Mol. Biol. PubMed Scopus Google Scholar, E. PubMed Scopus Google Scholar, P. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). 32 a and polymerase by structure in the T4 polymerase is not of strand synthesis. synthesis of both the leading and lagging strands the 32 and in to gene 59 protein several in to fork DNA Nossal N.G. J. Mol. Biol. PubMed Scopus Google Scholar, Nossal N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar) and in the DNA J. Alberts B.M. J. Biol. Chem. Full Text PDF Google Scholar, P. Nossal N.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The 59 protein but with that of 32 protein J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In vitro replication is in the of 59 protein of the of the In this study a to that in K. Z. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with the T7 replication proteins. M13 circular with a fork with the highly purified T4 replication and the of the DNA and DNA-protein complexes engaged in replication examined by the of a trombone loop in the T4 system and the that the segments in Okazaki fragment synthesis are organized into compact and and T4 DNA obtained from and and from The bacteriophage T4 gene 32 DNA 59 and proteins purified to as N.G. P. PubMed Scopus Google Scholar). proteins of The and proteins from the proteins the of T4 in E. DNA and of the circular duplex with a fork, and DNA synthesis with the purified T4 proteins are Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). with the and the 32 and 59 proteins for at synthesis by of a of polymerase and and from to of the with of to the synthesis. for by by with and at a of and at for of with 10 and then for as of to the the with for at and then of with 10 The to and with at PubMed Scopus Google Scholar). with E. coli protein K. Z. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and for as examined in a at by the with a to the and using for with a and the and arranged using of by the T4 a of the synthesis of DNA fragments in a circle replication by the purified T4 replication proteins. This to the reactions for and to of the fragments by but in the reactions for The for the and is circle to which is the of the are to the a tail. the has been this tail is to strand by the T4 replication P. Nossal N.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). To the synthesis of DNA the DNA for with the T4 32 59 and as In the of polymerase with the and 32 protein synthesis of the DNA circle within the and leading strand synthesis by the kb the are lagging fragments the is both and are for synthesis. In is 59 In the of the leading strands are at the that not at there are leading strands and lagging strand that the has In the with 59 protein the is as as the circle is at there are leading strand large to been and there are lagging strand In the of the is by the 59 but there are lagging strand that synthesis with 32 protein the of the of both and T4 the and of the DNA from the of the Okazaki fragments M. Nossal N.G. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In the presence of T4 and a of the Okazaki lagging strand fragments are as by the of lagging strand and the in The kb is circular from molecules that not strand synthesis. of of the DNA in circle replication reactions the structure of the and pattern of and segments the replicating tail can be by the segments of molecules are extended and by E. coli protein J. T. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Z. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). are in and to stages of Okazaki fragment synthesis for trombone loop lagging strand is the lagging strand by the coordinated of the the T4 gene the a replication loop is the lagging strand DNA polymerase to and of the The replication loop has both and as lagging strand synthesis the of the portion of the loop the of the The in the loop to in as the at the fork the the lagging strand polymerase the fragment the of the fragment and then the trombone loop as in is not the for the next fragment is the loop is This model is from the trombone model proposed by Alberts (1Sinha N.K. Morris C.F. Alberts B.M. J. Biol. Chem. 1980; 255: 4290-4293Abstract Full Text PDF PubMed Google Scholar, 2Alberts B.M. Barry J. Bedinger P. Formosa T. Jongeneel C.V. Kreuzer K.N. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 655-668Crossref PubMed Google Scholar, 3Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google reactions with the and the T4 replication proteins and at of of DNA that of the circles been to fully circles of circles and and circles of the circles tails that 100 kb as by The tails fully duplex with the of one segments at the fork that of The tails highly coupled strand fully tails The pattern of segments to the fork can be to the stages of Okazaki fragment synthesis at the synthesis of a circle with one the circle from a fully tail to that replicating the Okazaki fragment and not synthesis of a fragment and a fragment to be and The portion DNA that by the the lagging strand fragment circles with into this of molecules with two segments the one the tail to the circle and the the tail from the by a These molecules in which the to the fork is generated by the is to by the synthesis of a Okazaki in which a tail to the rather the of the replication segments and the fork. These are not one Okazaki fragment is at a and their is The same for reactions in which the of and in which the of both and polymerase the replicating molecules for the of molecules with to segments the replicating tail by these in replication of segments a circle in a the lagging strand is into a trombone then the number and of the and segments at the fork in molecules which the trombone loop would be to the size of that would be at the fork the proteins are in In of this these for DNA from replication reactions the of replication proteins molecules containing one the in of the into the for a Okazaki fragment The the fragment there to be within of the molecules with two segments by one the of the the from the fork to the Okazaki fragment size the In molecules with segments the trombone the to the fork and the and This would be the to the fork Okazaki fragment that the completed. of and a at the T4 from in vitro replication reactions with the T4 proteins been by Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, Barry J. M. M. Alberts B.M. DNA and Scholar), the DNA-protein complexes by the proteins in to by not been To replication reactions for using the of proteins by to and for Examination of of replicating molecules several different DNA with a mass (replication of the molecules of circles that in the of into and fully duplex but a circle replication tail. of the DNAs of circles with a fully tail a single mass bound the circle These tails to 100 kb in In a of circles a replication complex and 56% one DNA and and the replication complex at the fork. The fully duplex and not contain segments of extended by 32 protein loop with the replication complex in the molecules The of the different species not by the of the to Okazaki the trombone the replication fork loop is generated by a of the lagging Because a loop be by the circle that the of the and the loop be the for a to be as containing a replication of the and molecules a the of circles with a replication complex and tail but and in the as circles with a tail by a single and a the of molecules that a single loop at the fork and molecules deproteinization, a by two segments and of loop for molecules containing one loop is in as as and these from the portion of the trombone then the in the of the segments the replication tail that by segments molecules as in the two are evidence that the from a folding of the lagging of the replicating molecules one loop at the fork, and two and The trombone not the presence of for molecules with segments the replicating tail Because the of these species are is that molecules with be to molecules with segments the circle tail. for these and segments are The into trombone the T4 and as the lagging strand loop of a and with the extended by the of 32 protein 32 to the for the to be the circle tail. to this the and tails fully duplex and in which there extended typical of is is bound by of 32 protein H. Alberts B. J. Mol. Biol. PubMed Scopus Google Scholar). Because molecules from the same reactions the pattern of and the segments must within the large replication complex at the fork. This is the replication complexes would be for a single complex of the T4 replication proteins the replication complexes a of in highly The replication complex would be to contain two of the T4 DNA polymerase and the with a molecular mass of is not the remains with the complex. The would contain a of and one of the and the 59 of the would be a size for the complex a of The mass of a of by a of molecules of 32 protein would be the full replication complex mass of To a mass for these which is by with the replication complexes and in which molecules to replication the the of the molecules and replication complexes and as in A. J. Mol. Biol. PubMed Scopus Google Scholar) The of the replication complex that of to a replication complex containing the full of replication proteins and of with 32 To the presence of in the replication a with In vitro replication with the T4 proteins with and Because 32 protein remains bound to in not be by a in in the replication Following a to 20 and and to 100 and The for to and then and for DNAs in which the replication complex and segments of by 32 protein and 59 protein from the of the replication complex These that the segments are in compact structures with the fork and replication proteins of synthesis. of the the T4 replication the for at with the the and 32 and 59 of a of and into the of the compact bobbins at the fork from the molecules to the of these proteins These circles and extended in not is the 32 the 32 and 59 proteins are with in the presence of these not This that the compact bobbins are not a of the of 32 and 59 proteins to replication be that J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) compact structures 59 and 32 proteins to M13 a of 59 protein that would replication To the for full replication reactions for to in the of 59 many of the circles not circle replication of the to and in those the in the replication complex and extended 59 the full replication reactions for in the of large of to be extended bound by 32 and 59 protein with circular templates These to a model in which the of the compact bobbins the presence of the full T4 replication machinery engaged in synthesis. The of the of this structure be in a In this the highly purified T4 replication proteins to the of the DNA strands and protein complex at a circle replication fork. circle with a fork as the by this in highly synthesis and of Okazaki fragments of a size in studies. that 64% of the templates to and circle replication as by the presence of a fully duplex a large replication complex at a single and a circle tail that fully duplex except for single-stranded segments the fork. single DNA loop from the lagging strand folding back with the replication complex in of the replicating major are the presence of these at the fork, for the T4 system but not demonstrated and the highly compact of the segments at the fork. This study a number of of in vitro DNA replication using the and T7 In study of T4 Barry J. M. M. Alberts B.M. DNA and Scholar) from reactions by but not of the T4 replication proteins In the of 59 protein not at that and not circle replication containing one two segments duplex tails and the of the and segments are in the same as and in Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Barry J. M. M. Alberts B.M. DNA and Scholar) three molecules with one two leading to that of lagging strand fragments is of the leading In of molecules with one two segments in with of of and molecules in This leading strand synthesis and of the next Okazaki fragment in the presence of 59 These the that in Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the same of proteins and to the of Okazaki fragments the circle of T4 polymerase to the Okazaki but the protein not the of Okazaki fragment synthesis. that the of fragments molecules as great as the fragment in the of The size of the fragments in the study is the same as there and the two The that the lagging strand at a fork back to the replication complex of the of replication and the of the T4 system (1Sinha N.K. Morris C.F. Alberts B.M. J. Biol. Chem. 1980; 255: 4290-4293Abstract Full Text PDF PubMed Google Scholar, 2Alberts B.M. Barry J. Bedinger P. Formosa T. Jongeneel C.V. Kreuzer K.N. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 655-668Crossref PubMed Google Scholar, 3Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google Scholar). to these for the of the at the replication fork into a size of to which to the major of the of the duplex of the Okazaki fragments in these reactions The in loop size is from a trombone in which the loop in the of the Okazaki fragment and the of a in of the containing a replication complex and Because that in the of replication with a loop be The of is a of the in the synthesis of Okazaki as to the in at the of a and synthesis of the next with a and duplex but that of the replication is in lagging strand synthesis. The of the T4 are to in J. J. Mol. Biol. PubMed Scopus Google Scholar, J. T. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Z. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) M13 circular templates with T7 replication proteins. In the T7 a of the M13 circles engaged in circle replication and of at the replication fork. The of loop for both templates for both templates and to the The of in the T7 study reflects the of a and in the of the T7 and T4 In of replication molecules that the one two and a of replication molecules with one loop The for these molecules is that a Okazaki fragment the fragment completed. that the of of the fragment to the fork is that of the and that the of the fragment is to that in molecules with a single fragment is with this the polymerase has from the fragment to a the synthesis of the polymerase has been from to the reactions polymerase and are not and the of molecules with one fragment not with in polymerase The that the segments are the of DNA by the to of T4 is this from fragment these replication N.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). by would not be to is that to by the polymerase for the segments with the polymerase the Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The that the of a fragment in the T4 system not been and that there is a in fragment size molecules Nossal N.G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). previously that the compact structure of the lagging strand of the and the is not the molecules with a single loop are a of the in vitro replication are a of In the trombone there are two segments at the fork. within the trombone loop and is the of of the fork. The the for the Okazaki fragment that is the trombone as the in the in size as the fragment In the both segments as extended 32 protein of 32 protein bound to in vitro H. Alberts B. J. Mol. Biol. PubMed Scopus Google Scholar, Alberts B.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the segments are not extended but rather in compact structures at the fork. These been termed bobbins the T7 K. Z. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mass by that the mass of the DNA-protein complex at the fork with a structure large to contain the replication and with the replication proteins with of with 32 protein in T7 studies. The T7 is in 32 protein E. coli and in the presence of the complexes are compact S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) as to 32 that extended the segments in the T4 would be arranged into bobbins The of T4 bobbins to these structures as of the machine that replicates and be the structure which the proteins are with this that the M13 circles in the T4 reactions in a extended by 32 protein and 59 protein as of 59 protein from the reactions to circle with segments that that the of the compact bobbins is the full replication this in The presence of a loop at the fork that there be at least a one of the proteins the lagging strand and a protein at the fork. This be the leading strand a 59 the by the In E. coli the two polymerase are within the III holoenzyme (4Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 5Wu C.A. Zechner E.L. Hughes Jr., A.J. Franden M.A. McHenry C.S. Marians K.J. J. Biol. Chem. 1992; 267: 4064-4073Abstract Full Text PDF PubMed Google Scholar, 6Maki H. Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6570-6578Abstract Full Text PDF PubMed Google Scholar, 7Glover B.P. McHenry C.S. Cell. 2001; 105: 925-934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). T4 DNA polymerase is a in N.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, PubMed Scopus Google Scholar, E. 2001; PubMed Scopus Google Scholar), there is Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google Scholar), and M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) evidence for T4 polymerase and evidence for T4 polymerase and 59 protein M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is that the of the lagging strand polymerase with the the This study has the at the of the proteins and DNA strands at a T4 replication fork generated by the full of T4 replication proteins. This of proteins of the in of E. coli and the has that be in of the complex that this study and with the T7 proteins is the size of Okazaki which the same the proteins the T7 the complex T4 and the a M13 circle a This of Okazaki fragment size the great in proteins to in the of the bobbins as the of Okazaki In as T4 DNA is the molecular of the DNA and proteins in the would T4 DNA molecules are produced within a single is that the of the segments at the fork into organized bobbins to the fork using the system into these E. for the T4 and for in the of the