Chapter 1

DNA Replication Initiation

Stephen J. Aves

Summary

DNA replication is fundamental to cellular life on earth, and replication initiation provides the primary point of control over this process. Replication initiation in all organisms involves the interaction of initia-tor proteins with one or more origins of replication in the DNA, with subsequent regulated assembly of two replisome complexes at each origin, melting of the DNA, and primed initiation of DNA synthesis on leading and lagging strands. Archaea and Eukarya share homologous systems for DNA replication initiation, but differ in the complexity of these; Bacteria appear to have analogous, rather than homolo-gous, mechanisms for replication initiation. This chapter provides an overview of current knowledge of initiation of chromosomal DNA replication in the three domains of life.

Key words: Origin of replication , Origin recognition complex , Pre-replication complex , Pre-initiation complex , MCM .

Apart from an initial contribution of about one metre of DNA from each of your parents at conception, you have synthesised the remaining 20–200 billion km of DNA in your body by the process of DNA replication. Researching DNA replication is important, not just for a knowledge of our own cell proliferation (and the sometimes fatal consequences if this doesn’t take place correctly), but also for an understanding of the propagation of life on this planet.

The basic principle underlying DNA replication was realised by Watson and Crick (1) : separation of the strands of the double helix and the synthesis of one daughter strand complementary to each according to the base pairing rules. The cellular machinery that carries out this task is similar in principle in all organisms, but there

1. Introduction

Sonya Vengrova and Jacob Z. Dalgaard (eds.), Methods in Molecular Biology, DNA Replication, vol. 521© Humana Press, a part of Springer Science + Business Media, LLC 2009DOI: 10.1007/978-1-60327-817-5_1

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are differences between the three domains of life – the Eukarya, the Archaea, and the Bacteria. In particular DNA replication in the Bacteria does not appear to be orthologous to that in the other two domains of life, for reasons that are not yet clear (2– 6) .

DNA replication is processive, yet it must clearly have a start and an end. Considering replication initiation, the cell must have machinery for establishing where and when to start replication, and how to co-ordinate it with other processes in the cell cycle. This overview will outline chromosomal DNA replication initia-tion in the three domains of life, concentrating initially on the eukaryotes.

Jacob, Brenner, and Cuzin in 1963 proposed the replicon model for initiation of DNA replication, based on ideas from gene expres-sion in bacteria, which postulated that a trans -acting factor (the ‘initiator’) binds to a cis -acting initiation site (7) . This is essen-tially true in all organisms, and the initiation site is now termed the origin of replication. In eukaryotes the ‘initiator’ is a six-subunit protein complex termed the origin recognition complex (ORC) (8) , although other viral or cellular systems have different ‘initiators’. The term replicon has survived and represents a unit of replication, i.e. the DNA which is replicated from a single origin of replication – in bacteria this corresponds to a single DNA molecule as each chromosome or plasmid has a single origin, but the much larger eukaryotic chromosomes possess many origins of replication and therefore each forms many replicons (9, 10) . Replicons can be detected individually by various single-DNA-molecule labelling and visualisation techniques ( see Chapters ‘Replication Initia-tion Point Mapping: Approach and Implications’, ‘ChIP-Chip to Analyze the Binding of Replication Proteins to Chromatin Using Oligonucleotide DNA Microarrays’, ‘Analyzing Origin Activation Patterns by Copy Number Change Experiments’, ‘Microscopy Techniques to Examine DNA Replication in Fission Yeast’ and ‘Incorporation of Thymidine Analogs for Studying Replication Kinetics in Fission Yeast’ in this volume).

The nature of an origin of replication has been the subject of much research. In eukaryotes, origins from different taxa differ in their properties, and the nature of origins also can vary between cells at different developmental stages within a single organism (reviewed in refs. 11 and 12 ). Despite this heterogeneity, differ-ent types of eukaryotic origin all bind to the heterohexamic ORC, which therefore serves to spatially define the sites of replication initiation in eukaryotic cells (13) . ORC comprises six related subunits

2. Marking Origins of Replication

DNA Replication Initiation 5

which are conserved in varying degree from yeasts to mammals. The chromatin-binding kinetics of ORC varies between organ-isms; ORC is bound throughout the cell cycle in budding yeast and fission yeast (14, 15) , but Orc1 is released from chromatin as mammalian cells progress into S phase (16, 17) .

Origins of replication have been best studied in yeasts. Assays for autonomously replicating sequences (ARSs) have revealed many DNA sequences which confer on plasmids the ability to replicate in the budding yeast Saccharomyces cerevisiae or in the fission yeast Schizosaccharomyces pombe (18, 19) . Two-dimensional gel electrophoresis and whole-genome origin mapping studies have confirmed that ARS sequences generally correspond to bona fide origins of replication, that the ‘strength’ of an ARS is corre-lated with its probability of initiating replication in the chromo-some, and that there are about 400 origins in each yeast genome with an average replicon size of ~35 kb. S. cerevisiae origins are typically short (100–200 bp) and are defined by sequence: they comprise an A domain containing a close match to the 11-bp ARS consensus sequence (ACS: A/TTTTATG/ATTTA/T) and one or more B domains which are AT-rich but do not have a specific consensus sequence. The ACS and some B elements bind ORC, and S. cerevisiae ORC subunits 1–5 are essential for binding (20) . Fission yeast origins are longer (0.5–1.5 kb) and comprise mul-tiple short (20–50 bp) AT-rich tracts which act synergistically to promote ORC binding. Fission yeast Orc4 possesses nine AT-hook domains for binding to these origins (21, 22) .

Vertebrate origins of replication are less well defined and may be developmentally regulated. Consistent with this low-resolution localisation, human and Drosophila ORC bind AT-rich DNA with no strong preference for any particular sequence. Zones of initia-tion have been identified at many loci, e.g. within a 55 kb inter-gene adjacent to the Chinese hamster dihydrofolate reductase (DHFR) gene, but initiation can frequently occur at very many sites within such zones (11, 12, 23) . Zones of initiation may span as little as 1 kb (e.g. human LMNB2 origin) or many hundreds of kilobases (mouse IGH origin in pre-B cells), and contain specific sequences essential for origin activity but no common sequence motifs (reviewed in ref. 12) . Chromatin status is also likely to be important for origin utilisation in eukaryotes: there is evidence for epigenetic cues, DNA topology, and transcription factors all playing a role in ORC recruitment (12, 24) . This is particularly the case in early animal embryos, in which a spacing mechanism appears to operate and there is no sequence specificity at all: for example regulated replication of any exogenous DNA will occur in cell extracts from Xenopus early embryos (25, 26) . This may be related to the need for rapid cell cycles in such tissues: at the mid-blastula transition origins of replication become more widely spaced and exhibit specific localisation.

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ORC marks origins of replication but does not form part of the replisome (the multi-protein complex that synthesises DNA at each replication fork). The first replisome component to be recruited to origins is Mcm2–7 (MCM stands for minichromosome mainte-nance, reflecting a yeast screen in which many of the genes were first identified (27) ). Mcm2–7 is a hexamer of related proteins which is thought to act as the core of the replicative helicase, unwind-ing DNA to provide single-stranded templates on which daughter strands can be synthesised according to the Watson and Crick base pairing rules. DNA replication is bidirectional from origins, so two (or more) Mcm2–7 complexes are loaded at each origin.

In contrast to other replisome components, the loading of Mcm2–7 on to chromatin occurs significantly in advance of rep-lication itself: during late mitosis and G1 phases of the cell cycle (28) . Mcm2–7 is loaded on to bound ORC mediated by two proteins, Cdt1 and the ORC-like Cdc6, to form the ‘pre-rep-lication complex’ (pre-RC) (Fig. 1 ). A G1-phase origin with a bound pre-RC is often said to be ‘licensed’ to replicate.

ATP binding and hydrolysis play a role in pre-RC formation. Cdc6 (in common with Orc1, 4, 5, and Mcm2–7) is an AAA + ATPase; binding of Cdc6 to ORC is ATP-independent but ATP hydrolysis by Cdc6 is required for Mcm2–7 loading (29) .

The presence of multiple origins on a chromosome provides redundancy and only a subset of these needs to give rise to repli-cation forks in order for the chromosome to replicate. Therefore individual licensed origins may or may not initiate DNA replica-tion in the subsequent S-phase; genome-wide analyses indicate that pre-RC-bound sites outnumber detectable replication origins (30) . On the other hand, no origin of replication should normally initiate more than once in the same cell cycle (31, 32) . This has been articulated in the Jesuit model (‘many are called but few are chosen’) (33) in which selection could be on the basis of chroma-tin status, metabolic conditions, or stochastic factors (34) .

Activation of the pre-RC requires two cell cycle triggers. These are the activities of two protein kinases: S-phase CDK (cyclin-dependent kinase) and Cdc7 kinase, also known as DDK (for Dbf4-dependent kinase). Phosphorylation events by these kinases lead to the recruitment at origins of a range of initiation proteins to form what is sometimes (again by analogy with tran-scriptional initiation) termed the pre-initiation complex or pre-IC

3. Formation of the Pre-replication Complex

4. Activation of the Pre-RC and Initiation of Replication

DNA Replication Initiation 7

(Fig. 1 ). Loading of further replisome components including DNA polymerases and melting of the DNA at the origin of repli-cation lead to formation of two replication forks and the start of DNA priming and synthesis (reviewed in ref. 35) .

Fig. 1. Initiation of eukaryotic chromosomal DNA replication. See text for details and references. pre-RC, pre-replication complex; pre-IC, pre-initiation complex; RPC, replisome progression complex; DDK, Dbf4-dependent kinase (Cdc7 kinase). The pre-RC forms prior to S-phase (late M, G1); activation of the pre-RC occurs at G1/S or during S-phase. Other RPC proteins include claspin (Mrc1) checkpoint mediator, fork protection complex (FPC), topoisomerase I, and the histone chaperone FACT. DNA polymerase ε may interact with TopBP1 at an earlier stage of initiation in S. cerevisiae . For clarity, DNA polymerase α -primase has been omitted from leading strands in the RPC .

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The identities of initiation proteins have become established over the last few years, although the exact mechanisms and order in which they interact is the subject of much research and may differ between organisms (35) . The experimental systems most frequently used are the budding yeast Saccharomyces cerevisiae , the distantly related fission yeast Schizosaccharomyces pombe , and Xenopus egg extracts. Origin binding is assayed in yeasts by chro-matin immunoprecipitation (ChIP) of origin sequences; asso-ciation of proteins with total chromatin has also been used. In Xenopus egg extracts replication initiation is sequence independent, so origin binding is assayed as ORC-dependent association of proteins with chromatin. Dependencies are generally established in Xenopus egg extracts by immunodepletion of specific compo-nents, and in yeasts by the use of temperature-sensitive mutants. Comparative conclusions from such studies can be difficult to reconcile as dissimilarities may be due to methodological differ-ences or alternatively reflect true variation between organisms.

Initiation proteins can be divided into two categories: those that form part of the replisome, and those that are required for initiation only and do not travel with the replication fork. In the former category are Mcm10, Cdc45, and the GINS com-plex (36) . Mcm10 is not part of the pre-RC and is unrelated to the Mcm2–7 proteins, but interacts strongly with them during S-phase (reviewed in ref. 37) . Mcm10 is required for Cdc45 load-ing (38– 40) , stimulates DDK activity in vitro, but loads in the absence of DDK in Xenopus (39, 41) . It is a zinc finger-containing protein with single-stranded DNA-binding activity that may indi-cate a role in origin melting, and it is required for the stability and activity of the initiating polymerase, DNA polymerase α -primase (reviewed in ref. 37) .

GINS is a complex of four small proteins (42, 43) (GINS stands for the Japanese for five, one, two, three, in reference to the individual names for these factors: Sld5, Psf1, Psf2, and Psf3). GINS and Cdc45 are thought to be cofactors for the helicase activity of Mcm2–7 at the replication fork: studies of the activity of these proteins in different systems variously refer to the CMG complex or the unwindosome (44, 45) . Cdc45 loading appears to be interdependent with that of GINS and to require the activity of Mcm10, DDK, and CDK (35) . More recent evidence indicates that GINS is dispensable for Cdc45 origin loading in budding yeast, but is required for its interaction with the active replisome (46) . In turn Cdc45 appears to be required for loading of RP-A (single-stranded DNA-binding protein), helicase activity, and loading of DNA polymerases (47, 48) .

Initiation proteins which do not travel with the replisome include TopBP1, Sld2, and Sld3 (36, 46) . Sld2 and Sld3 are CDK substrates and their phosphorylation leads to TopBP1 binding and origin association (49– 52) . TopBP1 is a BRCT repeat-containing

DNA Replication Initiation 9

protein which suffers from many aliases (TopBP1 in humans; Drosophila Mus101; Xenopus XMus101; S. cerevisiae Dpb11; S. pombe Rad4 or Cut5). TopBP1 and GINS loading are mutually interdependent in S. cerevisiae and S. pombe (43, 53) . In Xenopus TopBP1 loading is required for Cdc45 loading but does not require Mcm2–7 or CDK, suggesting a pathway independent from Mcm2–7/Mcm10 (54, 55) . In addition to its essential require-ment in the initiation of DNA replication, TopBP1 also has a well-characterised checkpoint role, but these two functions appear to be distinct and separable (56) . As well as binding to TopBP1, Sld3 can associate with Cdc45 (57, 58) . Cdc45 and GINS loading are interdependent with that of Sld3 in budding yeast but may be dependent on Sld3 function in fission yeast (53, 58) .

Much remains to be resolved concerning interactions and the roles of initiation factors in assembly of the replisome. DNA polymerase α -primase is the initiating polymerase and Mcm10 is required for its stability and its loading on to chromatin (59– 61) . Cdc45 can also facilitate loading of DNA polymerase α -primase on to origins of replication (62– 64) . Mcm10 also contains a PIP (PCNA-interacting protein) box and diubiquitinated forms of Mcm10 bind to PCNA, the sliding clamp for processive DNA polymerases δ and ε (65) . DNA polymerase α is required for PCNA recruitment in Xenopus (47) . DNA polymerase ε can syn-thesise leading-strand DNA in budding yeast (66) , and there is evidence in this organism for its association with initiating factors TopBP1 and GINS (67– 70) .

To summarise: initiation of DNA replication in response to CDK and DDK requires at least nine initiation factors including Mcm10, Cdc45, the GINS tetramer, TopBP1, Sld2, and Sld3. The exact functional interrelationships between these proteins are the subject of active research and may differ in detail between organisms, but they facilitate the assembly of RP-A and the three replicative DNA polymerases, and priming and initiation of DNA synthesis. Less still is known about the recruitment and assembly of regulatory proteins which associate with Mcm2–7 and GINS in the replisome progression complex (RPC), such as the histone chaperone FACT, the fork protection complex (FPC), the claspin (Mrc1) checkpoint mediator, and topoisomerase I (36) .

Sld2 and Sld3 appear to be the only CDK substrates required for initiation of DNA replication, at least in budding yeast (51, 52) . Mcm2–7 subunits are substrates for DDK (71, 72) ; Mcm2 is phosphorylated in cell-free replication systems and at G1/S in vivo.

5. Controlling the Initiation of Replication

5.1. CDK and DDK Substrates

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Vertebrates also possess an early embryonic Dbf4-related factor, Drf1, which is essential in Xenopus for Mcm4 phosphorylation, Cdc45 loading, and DNA replication (73) . Drf1 declines after gastrulation and Dbf4 increases.

DNA replication initiation is triggered by action of the CDK and DDK protein kinases on the pre-RC. This occurs as cyclin B-CDK activity increases late in G1 phase of the cell cycle (28) . Reinitiation at individual origins must be prevented until after mitosis, other-wise partially re-replicated DNA would result. Cells therefore have multiple mechanisms to prevent pre-RC formation in S/G2/early M phases of the cell cycle; these vary in detail between organisms and include transcriptional downregulation, proteolysis, nuclear exclusion, inhibitory phosphorylation, or cyclin binding of vari-ous essential pre-RC components (ORC/Mcm2–7/Cdc6/Cdt1) (14, 74– 79) . All of these mechanisms occur as a result of CDK activity thus providing a molecular switch: CDK activity initiates DNA replication but prevents further pre-RC formation; when CDK activity is lost at the metaphase-anaphase transition, pre-RC formation can occur but initiation is not possible. There are also CDK-independent mechanisms for inhibiting pre-RC formation from S-phase onset, for example PCNA-dependent degradation of Cdt1 (80, 81) , or geminin inhibition of Cdt1 in vertebrates (82) .

Not all replication origins are initiated simultaneously (83) . Ori-gins exist which fire reproducibly early or late in S-phase (84, 85) although chromatin context is also important (86, 87) : transcrip-tionally active DNA tends to replicate early in S; heterochromatin and gene-poor regions late (88) . Microarray methods show that neighbouring origins tend to initiate at the same time in S phase (30, 32, 89) . Early replication is correlated with histone acetyla-tion (90– 92) and, at least in mammals, with intranuclear position of the DNA (93, 94) . Developmental regulation of replication timing can also occur, for example at the murine immunoglobulin heavy chain (IgH) locus (95) and more generally in early embryos, e.g. Xenopus (26) . The mechanisms regulating the timing of ori-gin firing within S-phase are not well understood (83, 96) .

Eukaryotic DNA viruses utilise host cell DNA replication factors for DNA synthesis, but override host cell controls over replication initiation. Usually this is achieved by a virus-encoded initiation factor. In the model SV40 system the initiator is large T-antigen (T-Ag), a multi-functional protein which recognises the single origin of replication on the SV40 circular DNA genome. T-Ag acts as the replicative helicase for SV40 DNA, facilitates loading of DNA polymerase α -primase, and recruits topoisomerase I (reviewed in ref. 97) . The structure of the helicase domain of

5.2. Only Once

5.3. Timing Differences Between Origins

5.4. Eukaryotic Virus DNA Replication Initiation

DNA Replication Initiation 11

T-Ag has been solved (98) and shown to be a double-hexameric helix which is thought to act by pumping double-stranded DNA towards the dimer interface (99) .

The DNA replication system in Archaea (archaebacteria) is related to that in the Eukarya but is less complex, which has prompted the study of Archaea as models for eukaryotic DNA replication (100) . Archaea possess one or more ORC/Cdc6 homologue(s) which bind to one or more origin sequences, often situated close upstream of ORC/Cdc6 gene(s) (101) . For example, Pyrococ-cus abyssi has a single AT-rich origin ( oriC ) containing repeated motifs and situated immediately upstream of a single ORC/Cdc6 gene. Sulfolobus solfataricus has three ORC/Cdc6 homologues and three identified origins, two close upstream of ORC/Cdc6 genes, but the third approximately 80 kb from the third gene. Therefore at least some Archaea have multiple origins of replica-tion, as for eukaryotic chromosomes, rather than the single repli-con pattern found in Bacteria. The S. solfataricus origins contain origin recognition boxes (ORBs) which possess a region of dyad symmetry with a GC-rich region asymmetrically adjacent (100) . ORBs are conserved in whole or in part in a diversity of other Archaea, although experimental confirmation of origin activity is not available in most species (102) . Archaeal origins therefore appear to be defined by sequence, as for those of bacteria and budding yeast.

The structures of two archaeal ORC/Cdc6 proteins have been determined and reveal a C-terminal winged-helix domain, conserved in sequence in archaeal ORC/Cdc6 and eukaryotic Cdc6 proteins, which is thought to bind DNA. Archaeal ORC/Cdc6 proteins are AAA + ATPases, and ATP binding and hydroly-sis are likely to be important in their replication initiation function (103) . No archaeal homologue of the eukaryotic Cdt1 protein has been identified.

Archaeal MCM helicases are AAA + ATPases which form homohexamers and interact with a two-subunit primase via a GINS complex. The GINS complex is a dimer of Gins23 and Gins15 (each orthologous to two eukaryotic GINS subunits) plus the RecJdbh protein, which has homology to bacterial DNA-binding RecJ. In contrast to most eukaryotic Mcm2–7 pro-teins, archaeal MCM double hexamers have clear 3 ′ –5 ′ helicase and DNA-stimulated ATPase activities in vitro (reviewed in ref. 103) . An electron microscopy structure of the Methanobacterium

6. Replication Initiation in Archaea

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thermoautotrophicum MCM has been determined, as has a crystal structure of the double-hexameric N-terminal region (104, 105) . Similarities of structure suggest that it may translocate along DNA in a similar manner to SV40 large T antigen.

Much less is understood about controls acting over the ini-tiation of DNA replication in Archaea than in eukaryotes, but homologues of most of the various eukaryotic initiation proteins are not apparent. Archaea exhibit a variety of single-stranded binding proteins (SSBs) which generally show more similarity to eukaryotic RPA than to bacterial SSBs. Primase in Archaea is dimeric and homologous to the small catalytic and large regula-tory subunits of eukaryotic primase; however, the archaeal pri-mase is not associated with DNA polymerase subunits as is the case for eukaryotic DNA polymerase α (5, 6) .

DNA replication in Bacteria (eubacteria) is analogous rather than orthologous to eukaryotic and archaeal DNA replication systems (106) . A single origin of replication per bacterial chromosome, oriC , is recognised by a conserved bacterial ‘initiator’: DnaA. Typically, multiple DnaA molecules bind co-operatively to mul-tiple boxes at the oriC locus, leading to a local deformation of the DNA and melting of the origin region, facilitated by bacterial chromosome proteins. The gene for DnaA is adjacent to the ori-gin in many species, and the two may be co-regulated (reviewed in ref. 106) .

The bacterial initiator protein, DnaA, is an AAA + ATPase but is not orthologous to eukaryotic or archaeal ORC/Cdc6 and has a C-terminal helix-turn-helix domain to bind DNA, not a winged helix. ATP binding and hydrolysis are important for loading DnaB, the bacterial helicase. This AAA + ATPase forms a homohexameric helicase but is not orthologous to eukaryo-tic/archaeal MCM helicases. Two DnaB hexamers are loaded per oriC, one for each replication fork. Loading of DnaB also requires binding to the DnaC protein, which is also an AAA + ATPase (107) . Bacterial single-stranded binding protein (SSB) contains an oligonucleotide/oligosaccharide-binding fold (OB-fold) and forms a homotetramer, in contrast to eukaryotic RP-A which is a heterotrimer also containing a total of four OB folds. DnaG is the primase in Escherichia coli : it is a monomer and contains a zinc-binding fold and a TOPRIM (topoisomerase-primase) fold. DnaA is released as replication starts and rebinds before the next round of replication.

7. Replication Initiation in Bacteria

DNA Replication Initiation 13

Timely and co-ordinated initiation of DNA replication is critical for correct and accurate transmission of the genome. DNA rep-lication initiation mechanisms are understood, at least in outline, for all three domains of life on earth. Less well understood are the regulatory mechanisms operating over initiation at origins, the molecular and functional interactions between initiation fac-tors, the interactions between initiation proteins and components of the replisome, and the extent of similarities and differences between species within each domain.

I would like to thank Karen Moore for helpful comments on this chapter and to apologise to the many authors whose work I have not been able to refer to because of space limitations in this outline review.

8. Conclusions

Acknowledgements

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