Holding on and letting go

JONATHAN

M.

FRIEDMAN

POLYMERASE

Holding

STRUCTURES

on and letting go

A new structure yields insight into the molecular basis for processivity during gene replication. Prokatyotic and eukaroyotic gene replication are similar in several of their steps [ 11. These include recognition of the origin of replication, unravelling of the short stretch of duplex DNA near the origin, priming - that is, the synthesis of a short strand of RNA to serve as a primer for DNA synthesis - formation of an initiation complex for polymerization, DNA synthesis and, finally, termination. In the past year, significant advances have been made in our understanding of the protein factors involved in some of these processes. A common theme that has emerged over the past several years is that each of these processes involves large multiprote$ complexes. They are the rule, for example, for DNA polymerases involved in gene replication. In contrast, DNA polymerases associated with gene repair, such as DNA polymerase I from Escherichiu coli, are simpler and may consist of a single subunit. Subunits of polymerases involved in gene replication have been isolated, genes cloned, and proteins overexpressed in a number of laboratories to determine the nature of the interactions between the subunits in these complexes and to determine the functional roles of each subunit in the overall process of gene replication. Although there are differences in some of the details, it has been found that similarities may be drawn between the functional roles of polymerase subunits from different organisms. The similarities among viral, prokaryotic and eukaryotic polymerases, noted by the laboratories of Stillman [ 21, Alberts [ 31, and O’Donnell [41, are listed in Table 1. A functional distinction between DNA polymerases associated with gene replication and those associated with jlexes ,.

iif,.anaLg&is ,I. ,.C’

Eukaryotic DNA-dependent

,+5l&cal

DNA

ATPase

RF-C (replication

Processivity

factor (profilerating

Addition to the

of nucleotides primer terminus

Binding to sinisle stranded DNA/helicase Unraveling of duplex DNA to allow initiation of replication Synthesis of a short RNA primer to initiate replication

296

polym,yrase

Pol

factor

‘from.

di,T\ierit

Bacteriophage

‘DN.&~pal~t&-&c ,..

T4

DNA

44/66 complex (Gene 44 protein Gene 66 protein) 45

polymerase

: involyed E.co/i

in g&e. : ,: ” DNA

y complex

,+Ji{a;ioti.’

polymer&’

(@,S’,z,x,~

ILI subunits)

+

protein

B subunit

pol

a

T4-DNA

polymerase

Core

polymerase

43

protein)

(cL,~,E subunits)

Gene

32

protein

SSB (single stranded binding protein)

helicase

Gene

41

protein

Helicase

associated subunits of pol a)

Gene

61

protein

Primase

factor

F-associated

,

nuclear

RF-A

(An activity with two DNA

ivity

(Gene

(replication Pol

DNA polymerase III consists of at least 10 subunits (LX, f3, y, 6, 6’, E, 4, z, x, $1 [51. The holoenzyme can be resolved into several subcomplexes. Subunits CL,8, and 8 form a complex, known as the core enzyme, that can catalyze ‘non-processive’ complementary nucleotide addition with a process&&y of 10-20 nucleotides per DNAbinding event. The heterodimer formed between subunit 01,the subunit that exhibits polymerase activity, and subunit E, the subunit exhibiting 3’-5’ exonuclease activity, is sufficient either for ‘non-processive’ nucleotide addition [6] or for ‘processive’ nucleotide addition in the presence of a preformed initiation complex [7]. The p subunit was the first subunit of DNA polymerase III to be associated with the function of processivity [8,9]. In the presence of duplex DNA, the ‘y complex’ [lo], ,.

Gene

PCNA cell antigen)

6 and/or

C)

a

DNA repair is that the former are often more processive - that is, once they latch onto a DNA substrate molecule they do not let go until many cycles of nucleotide adcltion have occurred (see [ 11, pp 494-496). As an example, DNA polymerase I from E. coli - an enzyme associated with DNA repair - is not very progessive: the ratio of the rate at which the DNA product/substrate is released to the rate of nucleotide addition is such that only about l&SO nucleotides are added each time DNA polymerase I binds to a DNA substrate. Compare that with DNA polymerase III from E. coli- an enzyme associated with gene replication. This enzyme can be highly processive, carrying out several thousand nucleotide additions for each DNA-binding event. The laboratories of Kornberg and McHenry showed that the functions of nucleotide addition and processivity were segregated between different protein subunits of DNA polymerase III.

A)

@

1992

Current

Biology

Fig. 1. Summary

of the proposed

intermediate

steps

in processive

which is formed by association between the y, 6, 6’, x and + subunits [ 111, plays a crucial role in the formation of an ‘initiation complex’ that is necessary for processive DNA replication. Formation of the initiation complex requires ATP hydrolysis [ll] and this ATPase activity has been associated with a complex between the y subunit and either the 6 or 6’ subunit of the y complex [12]. The z subunit causes added processivity and may substitute for the y subunit in the formation of the preinitiation complex [4]. This 7 subunit confers upon the processive replication complex the ability to plow through regions of ,duplex DNA in the path of the growing primer strand and through regions of DNA secondary structure in the template strand [ 1 1] . ijphy is the initiation complex necessary for processive DNA synthesis by DN,A polymerase III? In other words, ihow do the proteins of the initiation complex interact with the processivity factor (j3 subunit) and with DNA? 1n analogy with much earlier work by Alberts and his col@rgues on the bacteriophage T4 replication system 131, ;Kornberg, O’Donnell and their collaborators, in a number of elegant experiments [5,14], have shown that the &subunit is a sliding clamp that binds firmly but non$&lically to DNA and that the y-complex performs the ,@itial ATF-coupled attachment of the p subunit to the DNA [14]. The j3 subunit of DNA polymerase III associates very tightly with DNA in the initiation complex but somewhat less tightly with the as heterodimer, which is $.$icient for polymerization. Thus, it was suggested [ 131 that processivity could be achieved by the association of the sliding clamp (p), already bound to DNA, with the c@reenzyme (a, 8, E) following a preliminary stage of attachment of the sliding clamp to DNA catalyzed by the y,complex (y, 8, F’, x, $) (Fig. 1). Earlier experiments ,171suggested th at d ecreases in the degree of processivity in the absence of an E subunit could arise from the exchange of single c1subunits between l3 subunits attached to different pieces of LINA. P’Donnell and colleagues [ I31 veriiied the sliding clamp model by showing that the y complex could load a number of p subunits onto circular DNA and that these subunits would remain attached as long as the DNA circle remained intact. Once the circles of DNA were opened up by a restriction endonuclease, the p subunits became dissociated from the DNA, suggesting that they slid off the ends of the lineariz.ed DNA product. In addition, the p subunits would not readily dissociate from linearized DNA if the free ends of the DNA were coated with protein

Volume 2

Number 6

1992

replication

of DNA

by DNA

polymerase

III.

- for instance, if both ends were single-stranded DNA coated with E. coli single stranded DNA-binding protein. Kuriyan and colleagues [ 151 have now exquisitely confirmed this sliding clamp model by determining the threedimensional crystal structure of the 0 subunit of DNA polymerase III. This structure, the first for a processivity factor, shows that the l3 subunit is doughnut shaped, with the diameter of the inner hole being about 35A (Fig. 2). As the hole is sufftcient to accommodate either A-form or B-form DNA, the l3 subunit appears to be capable of encircling DNA entirely. This mode of DNA binding is concordant with calculations showing the presence of a small ring of positive electrostatic potential on the surface of the inner hole. The only published related structure is that of a proteolytic fragment of DNA polymerase I [ 161, a non processive DNA-polymerase. In that case, it is likely that DNA binds in a cleft which is somewhat smaller than that observed for the J3subunit. Recent refinement of the structure has shown that part of this cleft is entirely encircled by protein (IS Beese and TA Steitz, personal communication).

Fig. 2. Schematic representation of the structure of the p subunit of DNA polymerase ill from E co/i in its role as a sliding clamp.

A further question one may ask concerning the sliding clamp model for processivity is how does the j3 subunit avoid binding specifically to particular DNA sequences? Kuriyan and his collaborators [15] show that the two monomers that comprise the l3 subunit are related by a two-fold symmetry axis that is perpendicular to the plane of the ring and passes through its center (Fig. 2). They suggest that the internal two-fold symmetry of the

homodimer ensures that if an a-helix from one monomer interacts with the major groove of duplex DNA, then the corresponding a-helix of the other monomer interacts with the minor groove on the opposite face of the DNA. The position of the accisof internal two-fold symmetry ensures that the two faces of the doughnut are different from one another, suggesting that only one of them interacts specifically with DNA polymerase III core enzyme. (The 0 subunit structure also has a pseudo-six-fold axis coincident with the true internal two-fold axis but out of phase with the radlial structure of DNA. Kuriyan and colleagues suggest that the resulting six-fold averaging of interactions between the protein and the DNA further dampens any specificity in the interaction.) An ancillary question is how are the two dimers glued together in the ring? First, there are small hydrophobic patches towards the center of each interface region. Kuriyan and his colleagues [15] calculated the electrostatic field due to the protein and showed that the amino terminus is associated with a small region of positive electrostatic potential whereas the carboxy terminus is associated with a region of negative electrostatic potential, suggesting that electrostatic interactions play a role in dictating the head-to-tail directionality of dimerization. Additionally, at the interface between the two monomers there are P-sheets that share p-strands from each monomer. Thus, electrostatics, hydrogen bonding, and hydrophobic interactions all seem to play important parts in the formation and maintenance of the dimer. How does the Struchlre of the p subunit of DNA polymerase III relate to those of two other processivity factors - the ‘proliferating cell nuclear antigen’ (associated with DNA polymerase 6 and E) from eukaryotes [17-191 and the bacteriophage T4 gene 15 protein [ 201. These replication factors are somewhat smaller than the p subunit. The presence of the pseudo-six-fold axis in the j3 subunit lead Kuriyan and colleagues [15] to propose that these other processivity factors, which are about twothirds the size of the p subunit, may form trimers, rather than dimers, with similar three-dimensional structures and pseudo-six-fold symmetry to the p subunit. This conclusion is consistlent with earlier native-state molecular weight determinaltions [ 21,221. How can circular duplex DNA be threaded through this circle of protein and how is the required ATP used? Is it the DNA circle or the protein circle that. must first be opened? These questions, and others, remain to be answered by biochemical and structural experiments of interactions between the subcomplexes of DNA polymerase III (y complex, the j3 subunit, the core enzyme) and DNA. References 1. 2.

3.

4.

298

KORNBERG A, BAKER TA: DNA Replication, 2nd edn. New York: W.H. Freeman; 1991. TSURIMOTO T, STIUAN B: Functions of replication factor C and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. E’roc Natl Acud Sci USA 1972, 87~1023-1027. MUNN MM, ALBERTS BM: The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction. J Biol Chem 1991, 266:20024-20033. O’DONNELL M, Sru~vwu PS: Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps. J Biol Chem 1990, 2!65:1173-1187.

5. MAKI S, KORNBERG A: DNA

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

polymerase III holoenzyme of Escherichia coli. III. Distinctive processive polymerases reconstituted from purified subunits. J Biol Cbem 1988, 263~65614569. MAIU H, KORNBERG A: Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits. Proc Nat1 Acud Sci USA 1987, 84:438+4392. STUDWELL PS, O’DONNEU M: Processive replication is conk,gent on the exonuclease subunit of DNA polymerase III holoenzyme. J Biol Chem 1990, 265:1171-1178. BURGERS PMJ, KORNBERG A, SAKAK&%A Y: The dnaN gene codes for the fl subunit of DNA polymerase III holoenzyme of Escbericbia coli. Proc Nat1 Acud Sci USA 1981, 78:5391-5395. JOHANSON KO, HAYNES TE, MCHENRY CS: Chemical characterization and purification of the p subunit of DNA polymerase III holoenzyme from an overproducing strain. J Biol Chem 1986, 261:11460-11465. MCHENRY CS, KORNBERG A: DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits. J Bzbi Cbem 1977, 252:647%6&i. MAKI S, KQRNBERG A: DNA polymerase III holoenzyume of EschericEa coli II. A novel complex including the y subunit essential for processive synthesis. J Biol Chem 1988, 263:6555-6560. ONRUST R, STLJKENBERG PT, O’DONNELL M: Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme. J Biol Cbem 1991, 266:21681-21686. STUKENBERG PT, STLJDWELLVAUGHAN PS, O’DONNELL M: Mechanism of the sliding jklamp of DNA polymerase III holoenzyme. J Biol Chem 1991, 266:11328-11334. O’DONNELL M: Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Eschericbia coli. J Biol Cbem 1987, 262:16558-16565. KONG X-P, ONRUST R, O’DONNJZLL M, KUR~VAN J: Three diensional structure of the p subunit of Eschericbia coli DNA polymerase IIl holoenzyme: A sliding DNA clamp. Cell 1992, 69425437. OLLIS DL, BRICK P, HAMLIN R, XUONG NC, STEI’IZ TA: Structure of large fragment of Escbeticbia coli DNA polymerase I with dTMP. Nature 1985, 313~762-766. BURGERS PMJ: Saccbaromyces cerevisiae replication factor C. II. Formation and activity of complexes with proliferating cell nuclear antigen and with DNA polymerases 6 and E. J Biol Chem 1991, 266~22698-22706. LEE S-H, KWONG AD, PAN Z-Q, HUR~TIZ J: Studies on the activator 1 protein complex,, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase 6. J Biol Cbem 1991, 266:594-602. LEE S-H, PAN Z-Q, KWONG AD, BURGERS PMJ, HUR~I?Z J: Synthesis of DNA by DNA polymerase E in vitro. J Biol Chem 1991, 266:22707-22717, NOSSAL NG, ALBERTS BM: Mechanism of DNA replication catalyzed by purified T4 replication proteins. In Bacteriophage T4. Edited by Mathews CK, Kutter FM, Mosig G, Berget RB. Washington DC: American Society for Microbiology; 1983:71-84. JARVIS TC, PAUL LS, VON HIPPEt PH: Structural and enzymatic studies of the T4 DNA replication system. I. Physical characterization of the polymerase accessory protein complex. J Biol Chem 1989, 26412703-12716. BA~JER GA, BURGERS PMJ: The yeast anaIogue of mammalian cyclin/proliferating cell nuclear antigen interacts with mammalian DNA polymerase 6. Proc Natl Acad Sci USA 1988, 85:7506-7510.

Jonathan M. Friedman, Department of Molecular Biophysics and Biochemistry, Yale University, 219 Prospect Street, New Haven, Connecticut 06511, USA. @ 1992 Current Biology