Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair

DNA ligase I belongs to a family of proteins that bind to proliferating cell nuclear antigen (PCNA) via a conserved 8-amino-acid motif [1]. Here we examine the biological significance of this interaction. Inactivation of the PCNA-binding site of DNA ligase I had no effect on its catalytic activity or its interaction with DNA polymerase beta. In contrast, the loss of PCNA binding severely compromised the ability of DNA ligase I to join Okazaki fragments. Thus, the interaction between PCNA and DNA ligase I is not only critical for the subnuclear targeting of the ligase, but also for coordination of the molecular transactions that occur during lagging-strand synthesis. A functional PCNA-binding site was also required for the ligase to complement hypersensitivity of the DNA ligase I mutant cell line 46BR.1G1 to monofunctional alkylating agents, indicating that a cytotoxic lesion is repaired by a PCNA-dependent DNA repair pathway. Extracts from 46BR.1G1 cells were defective in long-patch, but not short-patch, base-excision repair (BER). Our results show that the interaction between PCNA and DNA ligase I has a key role in long-patch BER and provide the first evidence for the biological significance of this repair mechanism.     

A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation

Proliferating cell nuclear antigen is best known as a DNA polymerase accessory protein but has more recently also been shown to have different functions in important cellular processes such as DNA replication, DNA repair, and cell cycle control. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin-dependent protein kinases and their regulatory partner, the cyclins. Here we show a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C termini. We found that PCNA and Cdk2 form a complex together with cyclin A. This ternary PCNA-Cdk2-cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of replication factor C as well as DNA ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA ligase I and to stimulate phosphorylation of DNA ligase I. Based on our results, we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an "adaptor" for Cdk2-cyclin A to PCNA-binding DNA replication proteins.     

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.     

A conserved interaction between the replicative clamp loader and DNA ligase in eukaryotes: implications for Okazaki fragment joining

The recruitment of DNA ligase I to replication foci and the efficient joining of Okazaki fragments is dependent on the interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA). Although the PCNA sliding clamp tethers DNA ligase I to nicked duplex DNA circles, the interaction does not enhance DNA joining. This suggests that other factors may be involved in the joining of Okazaki fragments. In this study, we describe an association between replication factor C (RFC), the clamp loader, and DNA ligase I in human cell extracts. Subsequently, we demonstrate that there is a direct physical interaction between these proteins that involves both the N- and C-terminal domains of DNA ligase I, the N terminus of the large RFC subunit p140, and the p36 and p38 subunits of RFC. Although RFC inhibited DNA joining by DNA ligase I, the addition of PCNA alleviated inhibition by RFC. Notably, the effect of PCNA on ligation was dependent on the PCNA-binding site of DNA ligase I. Together, these results provide a molecular explanation for the key in vivo role of the DNA ligase I/PCNA interaction and suggest that the joining of Okazaki fragments is coordinated by pairwise interactions among RFC, PCNA, and DNA ligase I.     

Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.     

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.     

Computational protein design suggests that human PCNA‐partner interactions are not optimized for affinity

Increasing the affinity of binding proteins is invaluable for basic and applied biological research. Currently, directed protein evolution experiments are the main approach for generating such proteins through the construction and screening of large mutant libraries. Proliferating cell nuclear antigen (PCNA) is an essential hub protein that interacts with many different partners to tightly regulate DNA replication and repair in all eukaryotes. Here, we used computational design to generate human PCNA mutants with enhanced affinity for several different partners. We identified double mutations in PCNA, outside the main partner binding site, that were predicted to increase PCNA‐partner binding affinities compared to the wild‐type protein by forming additional hydrophobic interactions with conserved residues in the PCNA partners. Affinity increases were experimentally validated with four different PCNA partners, demonstrating that computational design can reveal unexpected regions where affinity enhancements in natural systems are possible. The designed PCNA mutants can be used as a valuable tool for further examination of the regulation of PCNA‐partner interactions during DNA replication and repair both in vitro and in vivo. More broadly, the ability to engineer affinity increases toward several PCNA partners suggests that interaction affinity is not an evolutionarily optimized trait of this system. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase delta (Pol delta). Interactions with POL31, the second subunit of Pol delta, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase alpha, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol delta interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol delta complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol delta localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.     

Functional roles of p12, the fourth subunit of human DNA polymerase delta

Mammalian DNA polymerase delta (pol delta), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol delta were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol delta containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol delta and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol delta activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol delta activity. This supports the hypothesis that the interaction between pol delta and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol delta interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol delta-PCNA complex as well as pol delta itself.     

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.     

Phylogenetic analysis of archaeal PCNA homologues

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages. Received: July 10, 2000 / Accepted: September 26, 2000     

The replication factory targeting sequence/PCNA-binding site is required in G(1) to control the phosphorylation status of DNA ligase I.

The recruitment of DNA ligase I to replication foci in S phase depends on a replication factory targeting sequence that also mediates the interaction with proliferating cell nuclear antigen (PCNA) in vitro. By exploiting a monoclonal antibody directed at a phospho-epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphorylated in a cell cycle-dependent manner. After dephosphorylation in early G(1), the level of Ser66 phosphorylation is minimal in G(1), increases progressively in S and peaks in G(2)/M phase. The analysis of epitope-tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires both the nuclear localization and the PCNA-binding site of the enzyme. Finally, we show that DNA ligase I and PCNA interact in vivo in G(1) and S phase but not in G(2)/M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre-replicative form of DNA ligase I.     

Identification of a novel binding motif in Pyrococcus furiosus DNA ligase for the functional interaction with proliferating cell nuclear antigen

DNA ligase is an essential enzyme for all organisms and catalyzes a nick-joining reaction in the final step of the DNA replication, repair, and recombination processes. Herein, we show the physical and functional interaction between DNA ligase and proliferating cell nuclear antigen (PCNA) from the hyperthermophilic Euryarchaea Pyrococcus furiosus. The stimulatory effect of P. furiosus PCNA on the enzyme activity of P. furiosus DNA ligase was observed not at low ionic strength, but at a high salt concentration, at which a DNA ligase alone cannot bind to a nicked DNA substrate. On the basis of mutational analyses, we identified the amino acid residues that are critical for PCNA binding in a loop structure located in the N-terminal DNA-binding domain of P. furiosus DNA ligase. We propose that the pentapeptide motif QKSFF is involved in the PCNA-interacting motifs, in which Gln and the first Phe are especially important for stable binding with PCNA.     

Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase delta.

The formation of a complex between DNA polymerase delta (pol delta) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase delta to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol delta is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means. p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol delta and provides evidence for a site on pol delta that is involved in the protein-protein interactions between PCNA and pol delta. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol delta interacts with PCNA.     

Stimulation of 3'-->5' exonuclease and 3'-phosphodiesterase activities of yeast apn2 by proliferating cell nuclear antigen

The Apn2 protein of Saccharomyces cerevisiae contains 3'-->5' exonuclease and 3'-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3'-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3'-->5' exonuclease and 3'-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.     

An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation

The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.     

A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA

DNA sliding clamps encircle DNA and provide binding sites for many DNA-processing enzymes. However, it is largely unknown how sliding clamps like proliferating cell nuclear antigen (PCNA) coordinate multistep DNA transactions. We have determined structures of Sulfolobus solfataricus DNA ligase and heterotrimeric PCNA separately by X-ray diffraction and in complex by small-angle X-ray scattering (SAXS). Three distinct PCNA subunits assemble into a protein ring resembling the homotrimeric PCNA of humans but with three unique protein-binding sites. In the absence of nicked DNA, the Sulfolobus solfataricus DNA ligase has an open, extended conformation. When complexed with heterotrimeric PCNA, the DNA ligase binds to the PCNA3 subunit and ligase retains an open, extended conformation. A closed, ring-shaped conformation of ligase catalyzes a DNA end-joining reaction that is strongly stimulated by PCNA. This open-to-closed switch in the conformation of DNA ligase is accommodated by a malleable interface with PCNA that serves as an efficient platform for DNA ligation.     

Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA

Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA) and compared its structure–function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA (ScPCNA) by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2-amino alcohol, a small-molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility.     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.