Archaeal eukaryote-like Orc1/Cdc6 initiators physically interact with DNA polymerase B1 and regulate its functions

Archaeal DNA replication machinery represents a core version of that found in eukaryotes. However, the proteins essential for the coordination of origin selection and the functioning of DNA polymerase have not yet been characterized in archaea, and they are still being investigated in eukaryotes. In the current study, the Orc1/Cdc6 (SsoCdc6) proteins from the crenarchaeon Sulfolobus solfataricus were found to physically interact with its DNA polymerase B1 (SsoPolB1). These SsoCdc6 proteins stimulated the DNA-binding ability of SsoPolB1 and differentially regulated both its polymerase and nuclease activities. Furthermore, the proteins also mutually regulated their interactions with SsoPolB1. In addition, SsoPolB1c467, a nuclease domain-deleted mutant of SsoPolB1 defective in DNA binding, retains the ability to physically interact with SsoCdc6 proteins. Its DNA polymerase activity could be stimulated by these proteins. We report on a linkage between the initiator protein Orc1/Cdc6 and DNA polymerase in the archaeon. Our present and previous findings indicate that archaeal Orc1/Cdc6 proteins could potentially play critical roles in the coordination of origin selection and cell-cycle control of replication.     

The relationship between dNTP pool levels and mutagenesis in an Escherichia coli NDP kinase mutant

Loss of nucleoside diphosphate kinase (Ndk) function in Escherichia coli results in an increased frequency of spontaneous mutation and an imbalance in dNTP pool levels. It is presumed that the imbalance in dNTP pool levels is responsible for the mutator phenotype of an E. coli ndk mutant. A human homologue of Ndk and potential suppressor of tumor metastasis, nm23-H2, can complement the mutagenic phenotype of an E. coli ndk mutant. Here, we show that the antimutagenic property of nm23-H2 in E. coli is independent of dNTP pool levels, indicating that dNTP pool imbalance is not responsible for the mutator phenotype associated with the loss of ndk function. We have identified multiple genetic interactions between ndk and genes involved in the metabolism of dUTP, a potentially mutagenic precursor of thymidine biosynthesis. We show that loss of ndk function is synergistic with a dut-1 mutation and synthetically lethal with the loss of thymidine kinase function. Our results suggest that Ndk prevents the accumulation of dUTP in vivo. Based on these results and biochemical studies of Ndk, we propose that the mutagenic phenotype of an ndk mutant is caused by excess misincorporation of uracil in place of thymidine combined with a defect in the uracil base excision pathway.     

Functional analysis of mutant and wild-type Drosophila origin recognition complex

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.     

Functional analysis of an olfactory receptor in Drosophila melanogaster

Fifty nine candidate olfactory receptor (Or) genes have recently been identified in Drosophila melanogaster, one of which is Or43a. In wild-type flies, Or43a is expressed at the distal edge of the third antennal segment in about 15 Or neurons. To identify ligands for the receptor we used the Gal4/UAS system to misexpress Or43a in the third antennal segment. Or43a mRNA expression in the antenna of transformed and wild-type flies was visualized by in situ hybridization with a digoxigenin-labeled probe. Electroantennogram recordings from transformed and wild-type flies were used to identify cyclohexanol, cyclohexanone, benzaldehyde, and benzyl alcohol as ligands for the Or43a. This in vivo analysis reveals functional properties of one member of the recently isolated Or family in Drosophila and will provide further insight into our understanding of olfactory coding.     

Mutational specificity and genetic control of replicative bypass of an abasic site in yeast

Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA polymerases (Pols). Here we determine the mutational specificity and the genetic control of translesion synthesis (TLS) opposite an AP site in yeast by using a double-stranded plasmid system that we have devised in which bidirectional replication proceeds from a replication origin. We find that the rate, the genetic control, and the types and frequencies of nucleotides inserted opposite the AP site are very similar for both the leading and the lagging DNA strands, and that an A is predominantly inserted opposite the AP site, whereas C insertion by Rev1 constitutes a much less frequent event. In striking contrast, in studies that have been reported previously for AP bypass with gapped-duplex and single-stranded plasmids, it has been shown that a C is the predominant nucleotide inserted opposite the AP site. We discuss the implications of our observations for the mechanisms of TLS on the leading versus the lagging DNA strand and suggest that lesion bypass during replication involves the coordination of activities of the replicative Pol with that of the lesion-bypass Pol.     

Transcriptional silencing functions of the yeast protein Orc1/Sir3 subfunctionalized after gene duplication

The origin recognition complex (ORC) defines origins of replication and also interacts with heterochromatin proteins in a variety of species, but how ORC functions in heterochromatin assembly remains unclear. The largest subunit of ORC, Orc1, is particularly interesting because it contains a nucleosome-binding BAH domain and because it gave rise to Sir3, a key silencing protein in Saccharomyces cerevisiae, through gene duplication. We examined whether Orc1 possessed a Sir3-like silencing function before duplication and found that Orc1 from the yeast Kluyveromyces lactis, which diverged from S. cerevisiae before the duplication, acts in conjunction with the deacetylase Sir2 and the histone-binding protein Sir4 to generate heterochromatin at telomeres and a mating-type locus. Moreover, the ability of KlOrc1 to spread across a silenced locus depends on its nucleosome-binding BAH domain and the deacetylase Sir2. Interestingly, KlOrc1 appears to act independently of the entire ORC, as other subunits of the complex, Orc4 and Orc5, are not strongly associated with silenced domains. These findings demonstrate that Orc1 functioned in silencing before duplication and suggest that Orc1 and Sir2, both of which are broadly conserved among eukaryotes, may have an ancient history of cooperating to generate chromatin structures, with Sir2 deacetylating histones and Orc1 binding to these deacetylated nucleosomes through its BAH domain.     

Drosophila P elements preferentially transpose to replication origins

The P transposable element recently invaded wild Drosophila melanogaster strains worldwide. A single introduced copy can multiply and spread throughout the fly genome in just a few generations, even though its cut-and-paste transposition mechanism does not inherently increase copy number. P element insertions preferentially target the promoters of a subset of genes, but why these sites are hotspots remains unknown. We show that P elements selectively target sites that in tissue-culture cells bind origin recognition complex proteins and function as replication origins. The association of origin recognition complex-binding sites with selected promoters and their absence near clustered differentiation genes may dictate P element site specificity. Inserting at unfired replication origins during S phase may allow P elements to be both repaired and reduplicated, thereby increasing element copy number. The advantage transposons gain by moving from replicated to unreplicated genomic regions may contribute to the association of heterochromatin with late-replicating genomic regions.     

Crystal structure of a near-full-length archaeal MCM: Functional insights for an AAA+ hexameric helicase

The minichromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. Whereas the eukaryotic complex consists of 6 homologous proteins (MCM2–7), the archaeon Sulfolobus solfataricus has only 1 MCM protein (ssoMCM), 6 subunits of which form a homohexamer. Here, we report a 4.35-Å crystal structure of the near-full-length ssoMCM. The structure shows an elongated fold, with 5 subdomains that are organized into 2 large N- and C-terminal domains. A near-full-length ssoMCM hexamer generated based on the 6-fold symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer shows intersubunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four unusual β-hairpins of each subunit are located inside the central channel or around the side channels in the hexamer. Additionally, the hexamer fits well into the double-hexamer EM map of mtMCM. Our mutational analysis of residues at the intersubunit interfaces and around the side channels demonstrates their critical roles for hexamerization and helicase function. These structural and biochemical results provide a basis for future study of the helicase mechanisms of the archaeal and eukaryotic MCM complexes in DNA replication.     

Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA–oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA–oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.     

Temporal control of late replication and coordination of origin firing by self-stabilizing Rif1-PP1 hubs in Drosophila

In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the midblastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. Here, we show that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a “community effect” counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. We propose a model in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins.

During a typical metazoan cell cycle, large genomic domains initiate their replication at distinct times in S phase (1). Cytological studies over 60 y ago revealed that DNA sequences in the compacted heterochromatin replicate later in S phase compared to euchromatin (2, 3). These early studies and recent detailed analyses revealed a complex program among late replicating domains, in which different domains initiate replication with a specific delay (4). Execution of this stereotyped schedule occupies much of the S phase and must finish before mitosis. Despite recent advances in genomic methods for profiling global replication timing (5), the basis of the timing control is not yet solved, and we do not know how multiple origins are coordinated to fire together especially within repetitive DNA sequences.The Drosophila embryo offers a unique setting in which to examine the control of temporal programing of replication. In the earliest nuclear division cycles, there is no late replication, closely spaced origins throughout the genome initiate replication rapidly at the beginning of interphase, and their simultaneous action results in an extraordinarily short S phase (3.5 min) (6, 7). Late replication is developmentally introduced during the synchronous blastoderm nuclear division cycles, first influencing pericentric satellite sequences that form a major part of metazoan genomes (over 30% in Drosophila) (8). Individual blocks of satellite DNA are typically several megabase pairs in length, each composed of a different simple repetitive sequence. During the 14th cell cycle at the midblastula transition (MBT), the ∼6,000 cells of the entire embryo progress synchronously through a temporal program in which the different satellites are replicated with distinctive delays (4), dramatically extending the duration of S phase.The initial onset of late replication during development provides a simplified context in which to define its mechanism, because numerous complex features associated with replication timing have not yet been introduced. For example, chromatin states can have major impacts on replication timing. Consistent with this, late-replicating satellite sequences are usually heterochromatic, carrying the canonical molecular marks of constitutive heterochromatin (histone H3 lysine 9 methylation and HP1). During initial Drosophila embryogenesis, the satellites lack significant levels of these marks, and they replicate in sync with the rest of the genome (4, 9). Surprisingly, the introduction of the delays in replication to the satellite sequences precedes a major wave of heterochromatin maturation in the blastoderm embryo (4, 9–11). Furthermore, in a Rif1 null mutant (Rif1^KO), the S phase of cycle 14 is significantly shorter, and the late replication of satellite sequences is largely absent even though HP1 recruitment appears normal (10). Thus, a Rif1-dependent program bears virtually full responsibility for the S-phase program at the MBT.Rif1 is a multifunctional protein with an evolutionarily conserved role in regulating global replication timing (12). In species from yeast to mammals, mutation or depletion of Rif1 disrupts genome-wide replication timing (10, 13–18). Studies in a variety of systems revealed several aspects of Rif1 function. Yeast Rif1 associates with late origins (15, 19, 20), while the Rif1 of both Drosophila and mammals binds broadly within large late-replicating domains (10, 13, 14, 21). Rif1 has a conserved motif for interacting with protein phosphatase 1 (PP1) (22), and mutations in the PP1-interacting motifs lead to hyperphosphorylation of MCM helicase in the prereplicative complex (pre-RC) and the disruption of global replication timing (17, 23–27). Rif1 itself also harbors many sites recognized by S-phase kinases, including CDK and DDK, near its PP1-interacting motifs. In yeast, both a Rif1 mutant with phosphomimetic changes at these phosphorylation sites and a null mutation of Rif1 partially restore the growth defect of DDK mutants (15, 24, 25). These data suggest an interplay of Rif1 and DDK, wherein DDK acts first upstream of Rif1 phosphorylating it to disrupt its interaction with PP1, thus lowering the threshold of S-phase kinase activities required for origin firing. Second, DDK acts downstream to directly phosphorylate pre-RC and trigger origin firing (28–30). However, how these various features of Rif1 and DDK functions are integrated over large genomic regions to provide a domain-level control of replication timing remains elusive.Studies in flies indicate that Rif1 has adopted a developmental role in governing the onset of the late replication program described above. During the early embryonic cell cycles, high Cdk1 and DDK activities jointly inhibit maternally deposited Rif1, promoting synchronous firing of origins throughout the whole genome to ensure completion of DNA replication during the short interphases (4, 10, 31). As the cell cycle begins to slow and oscillations in Cdk1 activity emerge, a transient Rif1-dependent delay in the replication of satellite sequences slightly prolongs S phase. When the embryo enters the MBT in cycle 14, abrupt down-regulation of Cdk1 more fully derepresses Rif1, which accumulates in semistable foci (hubs) at satellite DNA loci (4, 10, 31, 32). High-resolution live microscopy reveals that different Rif1 hubs disperse abruptly at distinct times, followed by proliferating cell nuclear antigen (PCNA) recruitment as the underlying sequences replicate (10). Mutated Rif1 that is nonphosphorylatable at a cluster of CDK/DDK sites fails to dissociate from satellite DNA and dominantly blocks the completion of satellite DNA replication before mitosis. Conversely, ectopically increasing CDK activity in cycle 14 shortens the persistence of endogenous Rif1 foci and advances the replication program (10). These findings suggest that each Rif1 hub maintains a local nuclear microenvironment high in Rif1-recruited PP1 that inhibits DNA replication, and that kinase-dependent dispersal of Rif1 hubs is required to initiate the replication of satellite sequences. If we understood what coordinates Rif1 dispersal throughout the large Rif1 hubs, this model could explain how firing of clusters of the underlying origins is coordinated and how replication of different satellites occurs at distinct times. However, the precise mechanisms controlling the dynamics of Rif1 hubs remain unclear.Since Rif1 can recruit PP1 and form phosphatase-rich domains in the nucleus, we hypothesized that localized PP1 counteracts kinase-induced Rif1 dissociation so that the Rif1 hubs are self-stabilizing. If this self-stabilization is communicated within each hub, a breakdown in self-stabilization would lead to a concerted collapse of the entire hub and allow origin firing throughout the associated satellite sequence. Our findings herein indicate that the opposing actions of phosphatase and kinase combined with communication within the hubs create a switch in which a large phosphatase-rich domain is stable until kinase activity overwhelms the phosphatase. We propose that for large late-replicating regions of the genome, recruitment of Rif1-PP1 creates a new upstream point of DDK-dependent regulation in which DDK triggers the collapse of the phosphatase-rich domain to create a permissive environment for kinase-induced firing of all previously repressed origins.     

Origin recognition complex harbors an intrinsic nucleosome remodeling activity

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)₂ tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

DNA replication is a vital life process for all cell types—bacterial, eukaryotic, and archaeal. While there are important differences among the replication proteins of the three domains of life, they mostly function in similar ways. All of them use an origin binding protein that acts with other factors to load two hexameric helicases onto DNA for bidirectional unwinding of the duplex, and thus the ability to simultaneously replicate both strands of the cellular genome (1–3). The eukaryotic origin binding protein is a heterohexamer referred to as the origin recognition complex (ORC) (4). The sequences of the Orc1-6 subunits are conserved from yeast to human, and several of the subunits contain an adenosine triphosphate (ATP)-binding AAA⁺ module as in the Escherichia coli DnaA initiator. Origins in the budding yeast Saccharomyces cerevisiae occur in 100- to 200-bp DNA regions known as autonomously replicating sequences (ARSs) (5–10). However, the existence of ARSs is limited to only some species of budding yeast. Origins of replication with defined DNA sequences are not known at this time to exist in other eukaryotes (1, 2).The special feature of a defined origin sequence in S. cerevisiae has facilitated extensive characterization of the mechanism of DNA replication initiation (1). ORC interacts with Cdc6, Cdt1, and the minichromosome maintenance protein complex (Mcm)2–7 heterohexamer to assemble a Mcm2-7 double hexamer (referred to here as MCM DH) onto DNA in G1 phase (1–3). The loaded MCM DH is the “licensing” factor for replication because it acts as the marker for origin firing in S phase (11). Specifically, the MCM DH is acted upon by several initiation factors to form 2 larger 11-subunit CMG (Cdc45/Mcm2-7/GINS) helicases (12, 13). The two CMG helicases are oriented toward and pass each other to unwind DNA, and recruit the replicative machinery to form bidirectional replication forks (14, 15). ORC and the many other factors required to license an origin and form bidirectional replication forks are conserved in all eukaryotes.The yeast ARS is AT rich, which is not favorable to nucleosome binding (16, 17). Indeed, chromatin immunoprecipitation sequencing (ChIP-seq) studies indicate that many ARSs have a nucleosome-free region (NFR) that expands in G1/S phase (10, 18–20). Presumably the nucleosomes are moved aside to make way for ORC-mediated MCM DH formation at origins in G1 phase, and for CMG formation in S phase. In vitro studies demonstrate that in the presence of saturating nucleosomes, the ARS is functional for replication initiation without need for classic nucleosome remodelers (21), indicating that the expansion of the NFR at an ARS site may be achieved intrinsically by the origin recognition and replication machinery.We have recently reported that ORC binding to nucleosomes facilitates the loading of MCM DHs onto DNA, regardless of the DNA sequence (22). In that study, we observed the loss of the fluorescently labeled histone signal after ORC–nucleosome interaction, but did not investigate further the source and mechanism of this observation as it was not the focus of the study. Considering that ORC binding is the first step of origin licensing and that ORC harbors multiple ATPase sites, here, we explored the possibility that ORC itself may possess an ATP-facilitated nucleosome remodeling activity. Using single-molecule fluorescence microscopy combined with optical trapping, we find that ORC is indeed an ATP-dependent nucleosome remodeler with the ability to eject H2A-H2B dimers. ORC-mediated nucleosome remodeling may represent the inaugural event toward creating a local chromatin environment permissive to replication initiation.     

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Clamp-loader complexes are heteropentameric AAA+ ATPases that load sliding clamps onto DNA. The structure of the nucleotide-free Escherichia coli clamp loader had been determined previously and led to the proposal that the clamp-loader cycles between an inactive state, in which the ATPase domains form a closed ring, and an active state that opens up to form a "C" shape. The crystal structure was interpreted as being closer to the active state than the inactive state. The crystal structure of a nucleotide-bound eukaryotic clamp loader [replication factor C (RFC)] revealed a different and more tightly packed spiral organization of the ATPase domains, raising questions about the significance of the conformation seen earlier for the bacterial clamp loader. We describe crystal structures of the E. coli clamp-loader complex bound to the ATP analog ATPgammaS (at a resolution of 3.5 A) and ADP (at a resolution of 4.1 A). These structures are similar to that of the nucleotide-free clamp-loader complex. Only two of the three functional ATP-binding sites are occupied by ATPgammaS or ADP in these structures, and the bound nucleotides make no interfacial contacts in the complex. These results, along with data from isothermal titration calorimetry, molecular dynamics simulations, and comparison with the RFC structure, suggest that the more open form of the E. coli clamp loader described earlier and in the present work corresponds to a stable inactive state of the clamp loader in which the ATPase domains are prevented from engaging the clamp in the highly cooperative manner seen in the fully ATP-loaded RFC-clamp structure.     

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O(8) atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.     

The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K⁺HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K⁺HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K⁺HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K⁺HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K⁺HSFs. After DNA damage, appreciably more foci were formed in 55K⁺HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K⁺HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability.