On the Processivity of DNA Replication

Proc. Natl. Acad. Sci. U.S.A.

1993

The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects ofthe elongation phase ofDNA replication. Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32). The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex. We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex. The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32. A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction. These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase. A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43.The past few years have seen a coalescence of mechanistic themes in DNA replication. This is especially true of the emerging role of the polymerase accessory proteins in forming a replication holoenzyme that is capable of elongating DNA in a highly processive manner. In T4, Escherichia coli, and many eukaryotic systems, the assembly of the functional holoenzyme appears to require hydrolysis of ATP by accessory protein subunits complexed with DNA (see ref. 1).The T4 holoenzyme contains the T4 DNA polymerase together with the gp44/62 ATPase complex, the gp45 protein, and the gp32 single-stranded DNA binding protein. The assembly of these components into a processive holoenzyme is driven by ATP binding and hydrolysis, although the precise molecular details of this process remain unknown (for recent reviews, see refs. 2 and 3). The functional role of the accessory proteins in establishing the holoenzyme appears to be to form a "sliding clamp" (4, 5) that holds the polymerase to the DNA template.Recent progress in understanding the E. coli DNA polymerase III holoenzyme system (for review see refs. 6 and 7) suggests how the T4 proteins might form such a complex. The E. coli ycomplex (which consists offive subunits) hydrolyzes ATP and, in so doing, transfers (3 subunits (as a dimer) onto the primer template DNA substrate. The ( dimer then interacts directly with the core DNA polymerase (8, 9) to form a processive DNA replication holoenzyme. The recent elucidation of the three-dimensional structure of the , dimer has elegantly revealed the molecular basis ofthis process; the (3 dimer forms a ring that topologically encircles the DNA, whereby it presumably clamps the E. coli polymera...

Section: Resultsmentioning

confidence: 83%

Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase.

Reddy

Weitzel

Proc. Natl. Acad. Sci. U.S.A.

1993

“…In this paper, we have shown that rapid and processive strand displacement leading-strand DNA synthesis can be achieved with a properly loaded and activated two-protein helicase-polymerase system. Yet it is also clear that under physiological salt conditions even minimal processive synthesis by polymerase alone on a single-stranded template requires participation of the gp44͞62 and gp45 accessory proteins (or their equivalents in other replication systems) to form and to load the sliding processivity clamp and thus to tether the polymerase (and the nascent DNA strand) onto the template (28)(29)(30)(31). More recently it has also been shown (26) that an essential role of the gp61 primase subunit is to equivalently tether and stabilize the hexameric gp41 helicase onto the DNA to form a primosome complex that is sufficiently stable to complete the replication of an entire T4 genome.…”

Section: Discussionmentioning

confidence: 99%

A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis

Dong

Weitzel

Proc. Natl. Acad. Sci. U.S.A.

1996

We have developed a coupled helicasepolymerase DNA unwinding assay and have used it to monitor the rate of double-stranded DNA unwinding catalyzed by the phage T4 DNA replication helicase (gp41). This procedure can be used to follow helicase activity in subpopulations in systems in which the unwinding-synthesis reaction is not synchronized on all the substrate-template molecules. We show that T4 replication helicase (gp41) and polymerase (gp43) can be assembled onto a loading site located near the end of a long double-stranded DNA template in the presence of a macromolecular crowding agent, and that this coupled ''twoprotein'' system can carry out ATP-dependent strand displacement DNA synthesis at physiological rates (400 to 500 bp per sec) and with high processivity in the absence of other T4 DNA replication proteins. These results suggest that a direct helicase-polymerase interaction may be central to fast and processive double-stranded DNA replication, and lead us to reconsider the roles of the other replication proteins in processivity control.The bacteriophage T4 DNA replication (elongation) complex is composed of seven phage-coded proteins (1, 2). The DNA polymerase (gene 43 product, gp43) plays the central role in replication, in that it catalyzes the template-directed incorporation of nucleotide triphosphates (dNTPs) into the nascent DNA in both leading-and lagging-strand synthesis. Under physiological conditions, the polymerase together with the three T4 polymerase accessory proteins (gp44͞62 and gp45) and the single-stranded (ss) DNA binding protein (gp32) interact to form a holoenzyme complex that is capable of rapid and processive DNA chain elongation on ssDNA templates. The addition of the T4 replication helicase (gp41) and primase (gp61) results in the formation of a seven-protein complex that can catalyze rapid, processive, and coupled leading-and lagging-strand DNA replication on double-stranded templates. The helicase and the primase work together to form a primosome subassembly within the replication complex, which is responsible for unwinding the double-stranded (ds) DNA template downstream of the replication fork and for producing and positioning pentameric oligoribonucleotides to prime the synthesis of Okazaki fragments on the lagging strand.Determinations of the unwinding properties of replication helicases have generally used an assay in which a prebound oligodeoxyribonucleotide that is complementary to part of a tailed DNA template is released from the template on the addition of ATP (e.g., see refs. 3-5). Such analyses can reveal helicase directionality (depending on where on the template the complementary oligomer is located) and how many DNA oligomers have been completely removed from their complementary templates. However, these assays cannot be used to determine how fast the helicase moves through the dsDNA in carrying out the unwinding reaction. Furthermore the ''product'' of such assays is, of course, simply two separated DNA strands that will spontaneously rehybridize unle...

“…We propose that this nonspecifically bound state of the DNA polymerase at the p-t DNA junction is thermodynamically similar to the binding state of the DNA polymerase on nonspecific DNAs (single-stranded or double-stranded), which (at physiological salt concentrations; see Ref. 42) represents a significantly less stable polymerase-DNA complex (K d ϭ 100 -240 nM; see top entries in Table II; see "Results").…”

Section: State 1 Represents a Nonspecific And Loosely Bound Interactimentioning

confidence: 91%

Function and Assembly of the Bacteriophage T4 DNA Replication Complex

Delagoutte

Journal of Biological Chemistry

2003

Self Cite

Complexes formed between DNA polymerase and genomic DNA at the replication fork are key elements of the replication machinery. We used sedimentation velocity, fluorescence anisotropy, and surface plasmon resonance to measure the binding interactions between bacteriophage T4 DNA polymerase (gp43) and various model DNA constructs. These results provide quantitative insight into how this replication polymerase performs template-directed 5 3 3 DNA synthesis and how this function is coordinated with the activities of the other proteins of the replication complex. We find that short (single-and double-stranded) DNA molecules bind a single gp43 polymerase in a nonspecific (overlap) binding mode with moderate affinity (K d ϳ150 nM) and a binding site size of ϳ10 nucleotides for single-stranded DNA and ϳ13 bp for double-stranded DNA. In contrast, gp43 binds in a site-specific (nonoverlap) mode and significantly more tightly (K d ϳ5 nM) to DNA constructs carrying a primer-template junction, with the polymerase covering ϳ5 nucleotides downstream and ϳ6 -7 bp upstream of the 3-primer terminus. The rate of this specific binding interaction is close to diffusion-controlled. The affinity of gp43 for the primer-template junction is modulated specifically by dNTP substrates, with the next "correct" dNTP strengthening the interaction and an incorrect dNTP weakening the observed binding. These results are discussed in terms of the individual steps of the polymerase-catalyzed single nucleotide addition cycle and the replication complex assembly process. We suggest that changes in the kinetics and thermodynamics of these steps by auxiliary replication proteins constitute a basic mechanism for protein coupling within the replication complex.Major cellular processes, such as DNA replication, DNA transcription, RNA translation, and protein degradation, are carried out by multiprotein assemblies (sometimes called macromolecular machines) (1), within which the constituent proteins act in a coordinated manner to perform the specific functions of the complex at issue. Furthermore, these protein machines interact within the cell and are functionally coupled to additional complexes. For example, in eukaryotes, the transcription machinery is coupled to the RNA processing complex (or spliceosome), which is itself coupled to the nuclear mRNA export system (2). Similarly, the rescue of a stalled DNA replication fork by recombination proteins illustrates the functional interactions that occur between the DNA replication, recombination, and repair assemblies (3). As a consequence, a coordination or coupling of protein activities exists, not only within a given multiprotein complex, but also between such complexes within the cell. Attaining a molecular understanding of this protein-protein coupling requires characterization of the activities and structures of the proteins as they function in isolation and also as they operate in the presence of one or more protein and/or nucleic acid interaction partners.The DNA replication machinery is a model of ...