Coupling of a Replicative Polymerase and Helicase: A τ–DnaB Interaction Mediates Rapid Replication Fork Movement  Sungsub Kim, H.Garry Dallmann, Charles S McHenry, Kenneth J Marians  Cell  Volume 84, Issue 4, Pages 643-650 (February 1996) DOI: 10.1016/S0092-8674(00)81039-9

Figure 1 Replication Forks Produce Shorter Leading and Lagging Strands in the Absence of τ (A) Standard rolling circle replication reactions with the omission of the indicated protein or proteins were performed and analyzed as described in Experimental Procedures. Complete replication forks synthesize two populations of nascent DNA: a long leading strand that barely enters the gel and migrates as a band at about 50 kb and a population of short Okazaki fragments that migrate as a smear between 5 and 0.6 kb. PP, primosomal proteins. In the lanes labeled DnaB and PP, both these proteins and τ were omitted from the reaction. DNA synthesis for these reactions were (left to right) 102.2 pmol, 14.4 pmol, 3.8 pmol, and 3.7 pmol of [32P]dAMP incorporated into acid-insoluble product. (B) τ is not required for rapid nascent chain elongation on primed poly(dT). DNA products formed in the presence of core and SSB and either the presence or absence of τ in a 2 s incubation starting from a β–poly(dT)–(dC40–dT40):5′-[32P]oligo(dG30) complex were analyzed as described in Experimental Procedures. Cell 1996 84, 643-650DOI: (10.1016/S0092-8674(00)81039-9)

Figure 2 Replication Forks Formed in the Absence of τ Move Very Slowly Replication fork rates were determined as described in Experimental Procedures using a β–TFII DNA complex in the presence of the preprimosomal proteins, SSB, and core, and in either the presence or absence of τ. DNA products were analyzed by alkaline agarose gel electrophoresis. Both the autoradiograms and lane profile obtained with a Fuji BAS 1000 phosphorimager are presented. (A and C) Replication forks formed with core and τ. (B and D) Replication forks formed with only core. Note the differences in time scale for the two types of forks. The length of the largest leading strand at each timepoint was determined by measuring the point of intersection between straight lines drawn through the background and the trailing (i.e., slowest moving) edge of nascent DNA. The TFII DNA preparation used in (B) had a higher percentage of dimer template than the one used in (A). There is always a fraction of the template that is inactive, yet still gets labeled by the addition of a limited number of nucleotides. This accounts for the apparent bands at about 7 and 14 kb. Cell 1996 84, 643-650DOI: (10.1016/S0092-8674(00)81039-9)

Figure 3 Rate of DNA Unwinding by the Preprimosome BamHI-digested 5′-[32P]TFII DNA was used as a substrate for unwinding catalyzed by the preprimosome as described in Experimental Procedures. Aliquots were removed at the indicated times from the start of the reaction, and the DNA products were analyzed without deproteinization by electrophoresis through neutral agarose gels. The presence of SSB bound to the single-stranded DNA accounts for the fuzzy nature of the bands. The arrow indicates the position of the 4 kb single strand (from the 5′ end of the TFII to the BamHI site) displaced by preprimosome-catalyzed unwinding. Cell 1996 84, 643-650DOI: (10.1016/S0092-8674(00)81039-9)

Figure 4 Processivity of Replication Forks in the Absence and Presence of τ (A) Complete replication reactions (lacking only primase) were incubated for 10 min at 30°C either in the absence or presence of the poly(dA):oligo(dT20) competitor (added at the beginning of the reaction). Incorporation of [32P]dAMP in the absence and presence of the challenge template was 22.1 pmol and 2.5 pmol, respectively. (B–D) Replication forks were formed in the presence of the β–TFII DNA complex, the preprimosomal proteins (PPP), SSB, core, and either the presence or absence of τ as described in Experimental Procedures. After a 1.5 min incubtion at 30°C, [α-32P]dATP and poly(dA):oligo(dT20), to 3 nM as 3′ ends of oligo(dT20), were added as indicated, and the reaction continued for 10 min. DNA products were analyzed as described in Experimental Procedures. (B) shows the autoradiogram of the gel, and (C) and (D) show the lane profile in the absence and presence of τ, respectively, as determined by phosphorimager analysis. Cell 1996 84, 643-650DOI: (10.1016/S0092-8674(00)81039-9)

Figure 5 τ and DnaB Form a Complex Complex formation between τ and DnaB was assessed by gel filtration chromatography as described in Experimental Procedures. (A) Protein profile: τ alone (closed squares); DnaB alone (open squares); a mixture of τ and DnaB (open circles); a mixture of τ and DnaB incubated in the presence of micrococcal DNase (closed squares). (B–D) SDS–PAGE analysis (13% gel): τ alone (B); DnaB alone (C); DnaB mixed with τ (D). Cell 1996 84, 643-650DOI: (10.1016/S0092-8674(00)81039-9)

Figure 6 Immunoblot of DnaB Interaction with HE Subunits DnaB interaction with HE subunits was detected by immunoblotting as described in Experimental Procedures with (A) or without (B) DnaB. The amounts of protein (in picomoles) listed to the left of the blots refer only to DnaB, all other proteins were present in the amounts indicated to the right of the blots. Cell 1996 84, 643-650DOI: (10.1016/S0092-8674(00)81039-9)

Figure 7 One Possible Arrangement of Proteins at the Replication Fork In this diagram, the leading- and lagging-strand polymerases are shown in an anti-parallel orientation. With this symmetry, only the τ subunit associated with the lagging-strand polymerase would likely be close enough to DnaB to establish a protein–protein connection. See text for details. The drawing is not to scale. Cell 1996 84, 643-650DOI: (10.1016/S0092-8674(00)81039-9)