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Enzymology and Mechanisms Zvi Kelman and Mike O'donnell

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Enzymology and Mechanisms Zvi Kelman and Mike O'donnell DNA replication: enzymology and mechanisms Zvi Kelman and Mike O'Donnell Cornell University Medical College, New York, USA Research into the enzymology of DNA replication has seen a multitude of highly significant advances during the past year, in both prokaryotic and eukaryotic systems. The scope of this article is limited to chromosomal replicases and origins of initiation. The multiprotein chromosomal replicases of prokaryotes and eukaryotes appear to be strikingly similar in structure and function, although future work may reveal their differences. Recent developments, elaborating the activation of origins in several systems, have begun to uncover mechanisms of regulation. The enzymology of eukaryotic origins has, until now, been limited to viral systems, but over the past few years, enzymology has caught a grip on the cellular origins of yeast. Current Opinion in Genetics and Development 1994, 4:185-195 Introduction In this review, we will focus on recent developments in the study of origin activation and the mechanism of The structure of duplex DNA is so simple and elegant action of chromosomal replicases. These areas encom- that one may have thought it would be simple to du- pass only a subset of the recent advances in the enzy- plicate. However, since the isolation of DNA polymer- mology of DNA replication. ase I first delivered replication mechanisms into the hands of enzymologists, the process has been shown to be far from simple. Over 20 proteins are utilized in Es- cherichia coil and probably more in higher organisms [1]. In fact, the individual functions of many of these Replication origins proteins are still unknown and even more proteins re- main to be identified. In overview, the process begins The first (and still the only) cellular origin to have at a specific sequence called an origin upon which been activated in vitro was oriC, the origin of the proteins bind and locally unwind the duplex, allow- E. coil chromosome [1]. Since that breakthrough, sev- ing invasion of a helicase. The helicase couples ATP eral phage and viral origins have been activated in hydrolysis to melt the duplex, and then the single- vitro. Origin activation requires multiple copies of stranded DNA (ssDNA) is coated with ssDNA-bind- an origin-binding protein and usually additional ori- ing protein (SSB). This SSB-ssDNA nucleofilament has gin accessory proteins, some of which have secondary no secondary structure and serves as the most efficient functions as transcriptional activators. As proposed by template for chromosomal replicases, multiprotein ma- Hatch Echols [2], the requirement for multiple proteins chineries characterized by their rapid and highly pro- in site-specific processes, such as activation of origins cessive DNA synthesis. One strand of the chromosome and promoters, is imposed by the need for a high fi- is synthesized continuously, but because of the antipar- delity of action on specific sequences embedded in the allel structure of the duplex, the other strand is copied bulk of chromosome DNA. These multiprotein-DNA discontinuously as a series of fragments (Okazaki frag- complexes are termed specialized nucleoprotein struc- ments). This discontinuous mode of replication neces- tures (snups) [2]. Origin initiation events in E. coli, sitates frequent reinitiation of DNA chains, which are phage ~ and simian virus 40 (SV40) are similar and primed by short RNA primers synthesized by a pri- appear to apply also to the more recently characterized mase. Most of the above proteins are thought to work systems of phage P1, bovine papilloma virus (BPV), together in one large replisome assembly for coordi- herpes simplex virus 1 (HSV-1), and yeast (see Fig. nated synthesis of both strands of the chromosome. 1). In most systems, the origin-binding protein is pre- Abbreviations ACS~ARS core consensus sequence; ARS.--autonomous replication sequence; BPV--bovine papilloma virus; HSV-l--herpes simplex virus 1 ; MCM--minichromosome maintenance; ORC---origin recognition complex; PCNA--proliferating cell nuclear antigen; pol/PoI~DNA polymerase; PP2A--protein phosphatase 2A; RF-C--replication factor C; RP-A--replication protein A; snup---specialized nucleoprotein structure; SSB~single-stranded DNA-binding protein; ssDNA--single-stranded DNA; SV40--simian virus 40. © Current Biology Lid ISSN 0959-437X 185 186 Chromosomes and expression mechanisms pared for origin activation and, for this review, we de- late the process is not yet clear. In stage III, the ori- fine this as stage I. Multiple copies of the origin-binding gin snup unwinds DNA within three AT-rich 13-mer protein then assemble onto the origin with the acces- repeats to form the open complex and this reaction sory proteins to form the snup (stage II). This untwists requires a supercoiled template [1]. Formation of the a section of AT-rich DNA to form an 'open complex' open complex is highly regulated [1]. Negative regula- (stage III), providing a point of entry for the helicase tion is achieved by the inhibitor of cellular initiation, (stage IV). After helicase entry, the chromosome can IciA, which competes with DnaA for interaction with be extensively unwound and replication forks can be the 13-mers and prevents the formation of the open assembled. Origin activation systems differ in the de- complex [7"°,8"°]. ATP also regulates the open com- tails of these steps and also in their regulation. In the plex formation. DnaA can bind either ATP or ADP following section, we will focus on these differences, tightly, but only the ATP form is active in unwinding with particular attention to recent developments. the 13-mers [11. The DnaA protein becomes inactive upon slowly hydrolyzing the ATP to ADP, and the ADP remains bound, thus preventing subsequent reinitia- Prokaryotic origins tions. The DnaA protein can be readily reactivated by Recent studies show that the activation of oriC is reg- acidic phospholipids, which catalyze the exchange of ulated at each stage (Fig. 1). In stage I, the DnaA ATP for the bound ADP [1,9"'], but only when DnaA origin-binding protein is inactive when bound to acidic is bound to or/C, this may underlie the early obser- phospholipids, but this repression can be relieved, and vations of replicon attachment to the membrane [10]. DnaA prepared for origin binding, by the DnaK heat RNA polymerase provides yet another level of reg- shock protein or by phospholipase treatment [3]. In ulation in determining the ability of R loops (RNA stage II, the origin snup is nucleated at four DnaA- paired with one strand of the DNA duplex) to pro- binding sites in oriC, upon which 20 or more DnaA mote the open complex, and although the transcript monomers assemble, along with several origin acces- need not enter on'C, it must be close [11]. In stage IV, sory proteins [1]. These accessory proteins include HU the DnaB helicase enters the open complex, but its ac- and IHF, which bend DNA, and they may act by help- cess absolutely requires the DnaC protein, a molecu- ing to wrap DNA around the DnaA subunits [4°',5]. lar matchmaker that delivers the hexameric DnaB into Other accessory proteins include Fis and the recently the origin in an ATP-dependent reaction, after which identified Rob protein [6"°]. They are not essential for DnaC departs from the DNA [1]. Origin unwinding is the activation of the origin, and how they may modu- bidirectional; therefore, two hexamers of DnaB must Stage I Stage I1 Stage III Stage IV DNA ~ r-f Open Prepare origin-binding Open complex Origin- protein ~ formation binding | I~ Replication fork System protein II + - II II + - II Helicase proteins E. coil DnaA DnaK Phospholipids HU. IHF, ATP ADP DnaB Dna(- Excess'~ Phospholipase Fis, Rob Acidic phospholipids IciA DnaC --| R loops / Dna(;-Prima~e Phage ~. ~. O HU RNA pol DnaB ~. p ~. p ~ SSB Dnal. DnaK ~ DNA po] III hoh,enzyme Gq)E Phage PI RepA DnaJ, DnaK, DnaA DnaB GrpE Yeast ORC ATP, ABFI -~ i)okx-Prin'=ase SV40 T-antigen Cdc2 psi ATP banligen RP-A(SSBI p53 ~ RP-A(SSB] PP2A Spl PP2A pol 8, t" (+RF-C, PCNA) BPV E1 E2 E! HSV-I UL9 UL5,8,52 ICP8 Helle ase--Primase(UL5,8.521 ICP8 (SSB) HSV-1 Pol q+LJt42) @1994 Currenl Opinion In Genetics and Developmenl Fig. 1. Regulation at different stages of origin activation in a number of systems. Origin activation has been subdivided into four stages: preparation of the origin-binding protein, assembly of the origin snup, local unwinding of origin DNA (the open complex), and entry of the helicase. Molecules that act on each stage are divided into positive (+) and negative (-) effectors and are shown for E. colt, phage )~, phage P1, yeast, SV40, BPV and HSV-1. A blank entry indicates that effectors of that step have not (yet) been identified. At the far right are listed the replication proteins needed to advance the replication fork. DNA replication: enzymolo~/and mechanisms Kelman and O'Donnell 187 be delivered to the open complex. Negative regula- the origin snup, is facilitated by ATP binding, which tion is achieved by the presence of too much DnaC effects the assembly of two hexamers of T-antigen protein, which binds to and inhibits the DnaB helicase onto the origin (reviewed in [25]). The two hexam- activity, possibly by preventing its translocation along ers are thought to encircle the origin DNA in SV40, DNA; therefore, a 'fine balance' of DnaC and DnaB is because if the hexamers are pre-assembled in solution essential to the productivity of this stage [12]. they do not bind the origin [26"']. Moreover, in elec- Phage ~. uses mainly host replication proteins for origin tron micrographs the double hexamer of T-antigen on activation, with the exception of the phage-encoded O the origin does not appear to wrap DNA around it as and P proteins, analogs of DnaA and DnaC [1].
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