Chapter 9: Replication of DNA Genomes

R DNA G Introduction Consequently, viral DNA synthesis cannot begin immediately upon arrival of the genome at the appropriate intracellular site, but rather is delayed until viral replication proteins have attained a sufficient concentration Replication of all DNA, from the genome of the simplest virus to that of the most complex vertebrate cell, follows a set of universal rules: (i) DNA is always synthesized by template-directed, stepwise incorporation of deoxynucleoside monophosphates (dNMPs) from deoxynucleoside triphosphate (dNTP) substrates into the 3' OH end of the growing DNA chain (ii) each parental strand of a duplex DNA template is copied by base pairing to produce two daughter molecules identical to one another and to their parent (semi conservative replication) (iii) replication of DNA begins and ends at specific sites in the template, termed origins and termini, respectively (iv) DNA synthesis is catalyzed by DNA dependent DNA polymerases, but many accessory proteins are required for initiation or elongation The genomes of RNA viruses must encode enzymes that catalyze RNA dependent RNA or DNA synthesis. In contrast, those of DNA viruses can be replicated by the cellular machinery In some cases, short RNA primers are first synthesized, as during replication of cellular genomes. In others, structural features of the genome or viral proteins provide primers Despite such distinctions, the replication strategies of different viral DNAs are based on common molecular principles and one of only two mechanisms: copying of both strands of a double-stranded DNA template at a replication fork or copying of only one strand while its complement is displaced Nevertheless, synthesis of these two DNAs is initiated by the same priming mechanism, and the herpesviral replication machinery carries out the same biochemical reactions as the host proteins that mediate synthesis of polyomavirus DNA DNA synthesis by the cellular replication machinery Eukaryotic replicons General features The replication of large eukaryotic genomes within the lifetime of an actively growing cell depends on their organization into smaller units of replication termed replicons Each replicon contains an origin at which replication begins. The sites at which nascent DNA chains are being synthesized, the ends of "bubbles" seen in the electron microscope, are termed replication forks The long-standing conundrum of how the second strand is synthesized was solved with the elucidation of the discontinuous mechanism of synthesis: RNA primers for DNA synthesis are made at multiple sites, such that the second new DNA strand is made initially as short, discontinuous segments, termed Okazaki fragments In chromosomal DNA, specialized elements, called telomeres, at the ends of each chromosome prevent loss of terminal sequences These structures comprise simple, repeated sequences maintained by reverse transcription of an RNA template, which is an essential component of the ribonucleoprotein enzyme telomerase Complete replication of all sequences of linear viral DNA genomes is achieved by various mechanisms, including continuous DNA synthesis from a DNA or protein primer Origins of cellular replication In contrast, this simple functional assay failed to identify analogous mammalian sequences, even when applied to DNA segments that included sites at which replication was known be initiated in cells Rather, initiation sites are defined by a variety of parameters, including proximity to active promoters, presence of CG-rich sequences, and chromatin structure Furthermore, genome-wide analysis of sites of both initiation of DNA synthesis and of binding of conserved replication proteins, such as the origin recognition complex (ORC), indicated that mammalian cells contain many more potential origins than are active during any one cell cycle Cellular replication proteins Eukaryotic DNA polymerases Mammalian cells contain several such nuclear enzymes, which are distinguished by their sensitivities to various inhibitors and their degree of processivity, the number of nucleotides incorporated into a nascent DNA chain per initiation reaction The requirements for viral DNA synthesis in vitro and genetic analyses identified DNA polymerases alpha, delta, and epsilon as the enzymes that participate in genome replication Only DNA polymerase alpha is associated with priming activity, because it is bound tightly to a heteromeric primase, which synthesizes short RNA primers Mechanisms of viral DNA synthesis Lessons from simian virus 40 The origin of SV40 DNA replication The SV40 origin was the first viral control sequence to be located on a physical map of the viral genome, in which the reference points were restriction endonuclease cleavage sites A short sequence, the core origin, which lies between the sites at which early and late transcription begin, is sufficient for initiation of DNA synthesis in infected cells Additional sequences within this busy control region of the viral genome increase the efficiency of initiation of DNA synthesis from the core origin Mechanism of SV40 DNA synthesis Origin recognition and unwinding The first step in SV40 DNA synthesis is the recognition of the origin by LT, the major early gene product of the virus When bound to ATP, LT assembles to form a double hexamer on the origin and elicits structural distortions in these flanking sequences In concert with cellular replication protein A (RP-A), which possesses single-stranded DNA binding activity, the intrinsic 3'-5’ helicase activity of LT then harnesses the energy of ATP hydrolysis to unwind DNA bidirectionally from the core origin Assembly of LT at the SV40 origin resembles assembly reactions at well-characterized bacterial origins, such as Escherichia coli OriC, or the origin of phage lambda, in which multimedia protein structures assemble on AT-rich sequences Leading-strand synthesis Binding of DNA polymerase alpha-primase to both LT and RP-A at the SV40 origin sets the stage for the initiation of leading-strand synthesis The 3’-OH ends of these fragments are then bound by cellular replication factor C (RF-C), proliferating - cell nuclear antigen (PCNA), and DNA polymerase delta This remarkable sliding clamp protein forms closed rings that track along the DNA template and serves as movable platforms for DNA polymerase Lagging-strand synthesis The first Okazaki fragment of the lagging strand is made by DNA polymerase alphaprimase, and the lagging strand is then synthesized by DNA polymerase delta Transfer of the 3' end of the first Okazaki fragment to this enzyme is thought to proceed as on the leading strand. The lagging strand template is then copied toward the origin of replication If the replication machinery tracked along an immobile DNA template, the complexes responsible for leading- and lagging-strand synthesis would have to move in opposite directions A more attractive alternative is that the DNA template is spooled through an immobile replication complex that contains all the proteins necessary for synthesis of both daughter strands Consistent with this idea, replication of chromosomal DNA occurs at fixed sites in the nucleus, and proteins that interact with the replicative helicase and both leading-strand and lagging-strand DNA polymerases have been identified LT is the helicase responsible for unwinding DNA at the origin, and remains associated with the replication forks, unwinding the template during elongation Termination and resolution Because the circular SV40 DNA genome possesses no termini, its replication does not lead to gaps in the strands made discontinuously These enzymes, which differ in their catalytic mechanisms and functions in the cell, reverse supercoiling, the winding of one duplex DNA strand around another In a closed circular DNA molecule, the unwinding of duplex DNA at the origin and subsequently at the replication forks is necessarily accompanied by supercoiling of the remainder of the DNA A single cycle of SV40 DNA synthesis produces two interlocked (catenated) circular DNA molecules that can be separated only when one DNA molecule is passed through a doublestrand break in the other Replication of chromatin templates The SV40 genome is organized by cellular nucleosomes both in virus particles and in infected call nuclei during transcription and replication However, numerous proteins that couple ATP hydrolysis to remodeling of nucleosomal DNA have been identified, and one shown to allow binding of LT to the origin and initiation of viral DNA synthesis in vitro Replication of other viral DNA genomes The replication of all viral DNA genomes within infected cells comprises reactions analogous to those necessary for simian virus 40 DNA synthesis, namely, origin recognition and assembly of a presynthesis complex, priming of DNA synthesis, elongation, termination, and often resolution of the replication products Synthesis of viral DNA molecules 1s initiated not only by RNA priming, but also by unusual mechanisms In which DNA or protein molecules function as primers Synthesis of viral RNA primers by cellular or viral enzymes As we have seen, cellular DNA polymerase alpha-primase synthesizes all RNA primers needed for replication of both template strands of polyomaviral genomes Viral primases can also synthesize RNA primers: this mechanism is characteristic of genome replication during productive infection by herpesviruses and probably poxviruses Priming via DNA: specialized structures in viral genomes Self-priming of viral DNA synthesis via specialized structures in the viral genome is a hallmark of all Parvoviridae, among the smallest DNA viruses that replicate in animal cells Adenovirus associated virus has a small (<5-kb) genome of single-stranded, linear DNA that carries inverted terminal repetitions (ITRs). Genomic DNA is of (+) and (-) polarity, for both strands are encapsidated, but in separate virus particles Formation of this structure at the 3' end of single-stranded viral DNA provides an ideal template-primer for initiation of viral DNA synthesis Following recognition of the free 3'-OH end of the viral DNA primer, the single template strand of an infecting genome can be copied by a continuous mechanism, analogous to leading-strand synthesis during replication of double-stranded DNA templates Adenovirus associated virus DNA synthesis is therefore always continuous and does not require DNA polymerase alpha-primase when reconstituted in vitro On the other hand, a specialized mechanism is necessary to complete replication of each strand, because the initial product retains the priming hairpin and is largely duplex DNA in which parental and daughter strands are covalently connected During this terminal resolution process, Rep 78/68 becomes covalently linked to the cleaved DNA at the sites that will become the 5' termini of the fully replicated molecules and singlestranded daughter genomes Following the synthesis of a duplex of the genomic DNA molecule (the replication intermediate), formation of the 3'-terminal priming hairpin allows continuous synthesis of single-stranded genomes by a strand displacement mechanism, with re-formation of the replication intermediate Rep 78 and Rep 68 are similar to simian virus 40 LT In several respects and can be considered origin recognition proteins In addition to recognizing and cleaving the terminal resolution site, these proteins provide the ATP-dependent, 3’—>5’ helicase activity needed for unwinding of the replicated ITR and re-formation of the priming hairpin Protein priming Initiation of DNA synthesis via a protein primer is a relatively rare mechanism, restricted to some bacteriophages and to hepadnaviruses and adenoviruses among those DNA viruses that infect animal cells The 5' ends of adenoviral genomes that enter infected nuclei are covalently linked to the terminal protein (TP), which is derived from a precursor during maturation of virus particles The 3’-OH group of the protein-linked dCMP then primes synthesis of daughter viral DNA strands by the viral DNA polymerase The structure of the DNA polymerase bound to its priming terminal protein suggests that such dissociation is the result of conformational change induced by displacement of the priming domain from the catalytic site in the polymerase Properties of viral replication origins Origins of replication are defined experimentally as the minimal DNA segment necessary for initiation of replication in cells or in vitro reactions Viral origins of replication support initiation of DNA synthesis by a variety of mechanisms, including some with no counterpart in cellular DNA synthesis Number of origins Different functions can be ascribed to the various Epstein-Barr virus origins: a single origin (OriP) allows maintenance of episomal genomes in latently infected cells, while the two others (OriLyt) support replication of the genome during productive infection The two types of origin possess considerable nucleotide sequence similarity but differ in their organization and can be distinguished functionally Viral replication origins share common features In general, AT base pairs contain only two hydrogen bonds, whereas GC pairs interact via three such bonds The less stable AT-rich sequences are thought to facilitate the unwinding of origins that is necessary for initiation of viral DNA synthesis on double-stranded templates Sequences adjacent to the polyomaviral and adenoviral core origins that increase replication efficiency include binding sites for transcriptional activators Assembly of viral permutation complexes on adenoviral origins is stimulated by direct interactions with cellular transcriptional activators that bind to adjacent sequences In other cases, such cellular proteins may promote viral DNA synthesis indirectly via alterations in the properties of nucleosides with which the viral genomes are associated Recognition of viral replication origins Properties of simian virus 40 LT Functions and organization The LT proteins of polyomaviroses provide functions essential for viral DNA synthesis, viral gene expression, and optimization of the intracellular environment Such host specificity is largely the result of species-specific binding of LT to the DNA polymerase alpha of the host cell in which the virus will replicate By binding and sequestering specific cellular proteins, LT perturbs mechanisms that control cell proliferation and can induce infected cells to enter S phase The properties of such altered proteins indicate that LT contains discrete structural and functional domains, such as the minimal domain for specific binding to the viral origin In the structure of a hexameric helicase domain assembled on Ori DNA with the AT-rich region unwound, the central channel is too narrow to accommodate double-stranded DNA Rather, interactions between local structural elements that line the channel and the DNA compress the sugar-phosphate backbones, leading to disruption of A-T base pairs and flipping out of the bases However, elegant studies of LT-dependent unwinding of single DNA molecules have established that LT translates along single-stranded DNA in the 3' —> 5' direction to unwind DNA by steric exclusion Regulation of LT activity Although production of LT is not regulated during the early phase of infection in simian cells in culture, its activity is tightly controlled Viral origin recognition proteins share several properties Other viral origin recognition proteins share with simian virus 40 LT the ability to bind specifically to DNA sequences within the cognate origin of replication The adenovirus-associated virus Rep 78/68 protein possesses these same activities but is also the site-specific endonuclease that is essential for terminal resolution Nevertheless, the E1 protein cannot support papillomaviral DNA replication in infected cells: a second viral protein, the E2 transcriptional regulator, is also necessary The E1 protein binds to origin DNA with only low specificity. In contrast, when the E1 and E2 proteins bind cooperatively, the specificity and affinity of the E1-DNA interaction are increased significantly Once an E1-E2 protein complex has assembled on the origin, hydrolysis of ATP bound to E1 appears to induce a conformational change that leads to dissociation of E2, allowing additional molecules of E1 to bind Viral DNA synthesis machines A particularly Simple viral replication apparatus is that of adenoviruses, which comprises the pTP primer, DNA polymerase, and only one other component, a single-stranded DNA binding protein Cooperative binding of this protein to single-stranded DNA stimulates the activity of the viral DNA polymerase as much as 100 - fold and induces highly processive DNA synthesis Rather, the DNA binding protein multimerizes via a C-terminal hook, and the formation of long protein chains by cooperative, high-affinity binding to single-stranded DNA provides the driving force for ATP independent unwinding of the duplex template Resolution and processing of viral replication products Replication of simian virus 40 DNA yields two interlocked, double-stranded, circular DNA molecules that must be separated by cellular topoisomerase II In other cases, replication yields multimeric DNA molecules, from which linear genomes of fixed length must be processed for packaging into virus particles It is well established that the linear viral genomes that enter infected cell nuclei at the start of a productive infection are converted rapidly to "endless" molecules in which the DNA termini are joined together This reaction requires cellular DNA ligase IV, which mediates joining of nonhomologous DNA ends during a cellular repair process, but it is not clear if unit-length circles or linear concatemers are produced Exponential accumulation of viral genomes Production of 1000 to 10,000 viral genomes, or more, per infected cell is not uncommon, as the products of one cycle of replication are recruited as templates for the next Viral proteins can induce synthesis of cellular replication proteins With few exceptions, virus reproduction is studied by infecting established cell lines that are susceptible and permissive for the virus of interest Many other cells in an organism divide rarely, or only in response to specific stimuli, and therefore spend much of their lives in G(0) Such cells lack many of the components of the replication machinery and are also characterized by generally low rates of synthesis of RNAs and proteins Functional inactivation of the RB protein Loss or mutation of both copies of the cellular retinoblastoma (rb) gene is associated with the development of tumors of the retina in children and young adults Because it is the loss of normal function that leads to tumor formation, rb is defined as a tumor suppressor gene In particular, the RB protein controls entry into the period of the cell cycle in which DNA is synthesized, the S phase, from the preceding (G1) phase Many of the genes that encode DNA polymerases, accessory replication proteins, and enzymes that catalyze synthesis of dNTPs contain E2F-binding sites in their transcriptional control regions As LT is the only viral protein required for viral DNA synthesis, its production seems likely to maximize the efficiency of genome replication, by coordinating initiation of this process with entry of the host cell into S phase A major consequence of activation of E2F In adenovirus-infected cells is stimulation of production of the three viral replication proteins The viral DNA polymerase, pTP primer, and DNA binding protein are encoded within the E2 gene, which is transcribed from an early promoter that contains two E2F binding sites In fact, these critical cellular regulators derive their name from their E2 binding sites in the E2 promoter, when were the first to be identified, and are necessary for efficient E2 transcription during the early phase The E1A-dependent regulatory mechanisms presumably operate synergistically to allow synthesis of the viral mRNAs that encode replication proteins in quantities sufficient to support numerous cycles of viral DNA synthesis Synthesis of viral replication machines and accessory enzymes In general, such proteins are dispensable for replication in proliferating cells in culture, because cellular enzymes supply the substrates for DNA synthesis However, herpes simplex viruses that lack thymidine kinase or ribonucleotide reductase genes cannot reproduce in neurons: such terminally differentiated cells are permanently withdrawn from the cell cycle and do not make enzymes that produce substrates for DNA synthesis Viral DNA replication independent of cellular proteins An alternative method to ensure replicative success of a DNA virus, regardless of the proliferation state of the host cell, is to encode all of the necessary proteins in the viral genome The genome of vaccinia virus, which is replicated in the cytoplasm, encodes a DNA polymerase, several accessory replication proteins such as the endonuclease required for resolution of the concatemeric products of DNA synthesis, and enzymes for synthesis of dNTPs Inhibition of cellular DNA synthesis When viral DNA replication is carried out largely by viral proteins, cellular DNA synthesis is often inhibited, presumably to increase the availability of dNTP substrates for viral genome replication There is some evidence that inhibition of cellular DNA synthesis is an active process rather than an indirect result of competition between viral and cellular DNA polymerases for the finite pools of dNTP substrates Synthesis of viral DNA is specialized intracellular compartments A common if not universal feature of cells infected by viruses with DNA genomes is the presence of virus-specific territories in which viral DNA synthesis takes place The replication of viral DNA genomes within infected cell nuclei also takes place in specialized compartments, which can be visualized as infected cell-specific foci that contain viral replication proteins and increase in size as new viral genomes are synthesized By definition, viral factories or replication centers contain viral DNA genomes delivered from infectious particles, newly synthesized viral DNA molecules, and the viral and/or cellular proteins needed for DNA synthesis The localization of the templates for viral DNA synthesis as well as the replication proteins to a limited number of sites undoubtedly facilitates efficient genome replication Viral factories and replication centers also serve as foci for viral gene expression, presumably in part by concentrating templates for transcription with the proteins that carry out or regulate this process, or that process primary transcripts Similarly, cytoplasmic vaccinia virus factories contain all the viral enzymes and other proteins necessary for synthesis of viral mRNAs and also host ribosomal and translation initiation proteins Another common property of nuclear viral replication centers is the presence of cellular DNA repair or recombination proteins The association of replication centers of different DNA viruses with constituents of the same intranuclear bodies suggests that reorganization of host cell nuclei facilitates viral DNA synthesis Limited replication of viral DNA genomes Integrated parvoviral DNA can be replicated as part of the cellular genome Although these helper viruses are widespread in hosts infected by adenovirus - associated viruses, the chances that a particular host cell will be infected simultaneously by two viruses are very low In fact, this is not the case, for this viral genome can persist by an alternative mechanism: in the absence of a helper virus, its genome becomes integrated into that of the host cell and is replicated as part of a cellular replicon In the absence of helper functions, Rep represses transcription from the promoter that controls its synthesis It is thought that double - stranded, circular genomes form initially, for example, upon annealing of complementary single-stranded genomes, and then undergo recombination to give rise to concatemers Different viral origins regulate replication of Epstein-Barr virus During herpesviral latent infections, the viral genome is stably maintained at low concentrations, often for long periods, and in some cases replication of viral and cellular genomes is coordinated Characteristic features of latent Epstein-Barr virus infection include expression of only a small number of viral genes, the presence of a finite number of viral genes, the presence of a finite number of viral genomes, and replication from a specialized origin Application of the episomal viral genome is limited to a few cycles. Subsequently, viral DNA genomes are duplicated once per cell cycle during S phase and partitioned evenly to daughter cells during mitosis However, this property cannot explain why each genome is replicated only once in each cell cycle, just as each cellular replicon: OriP and cellular origins fire once and only once in each S phase. Several mechanisms ensure that the essential MCM helicase is available only at the G1-to-S phase transition, and hence limit origin firing to once per cell cycle Direct observation of episomal viral genomes by in situ hybridization has established that these DNA molecules become tethered to cellular sister chromatids that are separated during mitosis In metaphase chromosomes, regions of less condensed (that is, accessible) AT-rich DNA are found between segments that are highly condensed As a latent infection is established, the Epstein-Barr virus genome becomes increasingly methylated at C residues present in CG dinucleotides These proteins [Zta and Rta] induce expression of the early genes that encode the viral DNA polymerase and other proteins necessary for replication from OriLyt In addition, Zta is the viral protein that recognizes the lytic origin (OriLyt) and Rta is necessary for replication from this origin Limited and amplifying replication from a single origin: the Papillomaviruses Papillomaviruses reproduce in the differentiating cells of an epithelium, with distinct modes of viral DNA synthesis associated with cells in various differentiation States Entry of a papillomaviral genome into the nucleus of an undifferentiated, proliferating basal cell initiates a period of amplification of the circular genome, just as during the early stages of latent infection by Epstein-Barr virus Replication continues until a moderate number of viral genomes (~50 to 100) has accumulated. A maintenance replication pattern, in which the viral genomes are duplicated on average once per cell cycle during S phase, is then established in basal wells The single viral origin and the viral E1 and E2 proteins that bind to specific origin sequences are necessary for both the initial amplification of the papillomavirus genome and its maintenance for long periods at a more or less constant concentration Stable maintenance of the viral genome requires an additional sequence, called the minichromosome maintenance element, which is composed of multiple binding sites for the E2 protein Remarkably, the final stage of papillomaviral DNA replication, production of high concentrations of the viral genome for assembly into progeny virus particles, is restricted to nondividing, differentiated epithelial cells, such as terminally differentiated keratinocytes Origins of generic diversity in DNA viruses Fidelity of replication by viral DNA polymerases Proofreading mechanisms Such fidelity, which is essential to maintain the integrity of the genome, is based on accurate base pairing during and after genome replication In such circumstances, the mismatched base in the primer strand is excised by a 3’—>5’ exonuclease present in all replicative DNA polymerases until a perfectly base-paired primer template is created Infection by these viruses (e.g., papillomaviruses and polyomaviruses) does not result in inhibition of cellular protein synthesis, and indeed may induce expression of cellular replication proteins Nevertheless, the rates of mutation of such genomes are considerably higher than those of double-stranded DNA genomes, on the order of 10^-6 substitutions/site/genome, and these viruses evolve rapidly Proofreading by viral DNA polymerases However, each of these viral enzymes possesses an intrinsic 3’->5’ exonuclease that preferentially excises mismatched nucleotides from duplex DNAs In vitro, and mutations that impair the exonuclease activity of the human adenovirus type 5 or herpes simplex virus type 1 DNA polymerase greatly increase the mutation rate Modulation of the DNA damage response Not surprisingly, such potentially lethal lesions elicit powerful DNA damage-sensing and response pathways that lead to DNA repair or, when damage is extensive, senescence or apoptosis of the affected cell The three DNA damage response pathways of mammalian cells are defined by the related protein kinases that transmit signals from the proteins that detect and bind to sites of DNA damage to effector proteins that both halt cell cycle progression and repair the lesions Replication of viral genomes can result In accumulation In infected cells of increased quantiles of single-stranded DNA (replication intermediates or viral genomes) or double-stranded DNA ends (linear viral genomes) In some cases, however, activation of these same repair pathways is necessary for amplification of DNA genomes and completion of the infectious cycle Inhibition of DNA damage responses Nevertheless, such inhibition is necessary not only to prevent ligation of replicated viral genomes into concatemers (which cannot be encapsidated) but also for efficient viral DNA synthesis: When neither the virus-specific E3 ubiquitin ligase nor the E4 Orf3 protein can be made in infected cells, viral genome replication is severely impaired, probably because NBS1dependent binding of MRN complexes to viral genomes precludes access to the origins of replication Differential impacts on DNA damage response pathways Herpes simplex virus 1 genomes enter infected cells as linear molecules with double-stranded ends that could trigger NHEJ and deleterious ligation of viral DNA molecules, as in human adenovirus-infected cells Despite inhibition of this process, activation of both the ATM and ATR pathways occurs in herpes simplex virus type 1 infected cells, and components of both pathways, including the kinases themselves, MRN proteins, and CHK1, are required for efficient genome replication and virus reproduction Active ATM phosphorylates LT at a specific residue, a modification required for efficient viral DNA synthesis In infected cells, and activation of both this and the ATR pathway prevents accumulation of stalled replication forks In viral DNA DNA damage responses essential for virus reproduction The viral E1 and E7 proteins induce activation of these signaling pathways and accumulation of activated ATM and proteins that mark sites of DNA damage, or that participate in homologous recombination at discrete nuclear foci that contain replicating viral genomes Recombination of viral genomes General mechanisms of recombination It [recombination] also makes a major contribution to repair of breaks in a DNA genome and can rescue replication when this process has stalled at unfavorable sequences in the template In site-specific recombination, exchange of DNA takes place at short DNA sequences that are specifically recognized by proteins that catalyze recombination, such as the lambda and retroviral integrates Much more common during reproduction of DNA viruses is homologous recombination, the exchange of genetic information between any pair of related DNA sequences Origin-independent, recombination - dependent replication In such recombination-dependent replication, recombination proteins catalyze the invasion of double-stranded DNA by a single DNA strand with a 3'-0H terminus, hence providing a primer for DNA synthesis Furthermore, herpes simplex virus DNA replication is accompanied by a high degree of recombination between repeated sequences in the genome Viral genome replication The integration of adenovirus-associated virus DNA into a specific region of chromosome 19, and its excision hen conditions are appropriate, is the result of site-specific recombination reactions mediated by the Rep 78/68 viral proteins, which bind to particular sequences in both viral and host DNA molecules Because the initial step in recombination, pairing of homologous sequences with one another, depends on random collision, it is concentration dependent Furthermore, the structures of replication intermediates, Or the nicking of viral DNA during replication or packaging that yields DNA ends, can facilitate recombination Recombination mediated by these viral proteins during infection is important for production of infectious genomes:the viral DNA synthesized and packaged in cells infected by mutants that do not direct synthesis of the active nuclease contains structural abnormalities and is poorly infectious