DNA Pol Research Articles

Potentiation of Temozolomide Cytotoxicity by Inhibition of DNA Polymerase β Is Accentuated by BRCA2 Mutation

Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.

The Transcription Elongation Factor NusA Is Required for Stress-Induced Mutagenesis in Escherichia coli

Stress-induced mutagenesis describes the accumulation of mutations that occur in nongrowing cells, in contrast to mutagenesis that occurs in actively dividing populations, and has been referred to as stationary-phase or adaptive mutagenesis. The most widely studied system for stress-induced mutagenesis involves monitoring the appearance of Lac(+) revertants of the strain FC40 under starvation conditions in Escherichia coli. The SOS-inducible translesion DNA polymerase DinB plays an important role in this phenomenon. Loss of DinB (DNA pol IV) function results in a severe reduction of Lac(+) revertants. We previously reported that NusA, an essential component of elongating RNA polymerases, interacts with DinB. Here we report our unexpected observation that wild-type NusA function is required for stress-induced mutagenesis. We present evidence that this effect is unlikely to be due to defects in transcription of lac genes but rather is due to an inability to adapt and mutate in response to environmental stress. Furthermore, we extended our analysis to the formation of stress-induced mutants in response to antibiotic treatment, observing the same striking abolition of mutagenesis under entirely different conditions. Our results are the first to implicate NusA as a crucial participant in the phenomenon of stress-induced mutagenesis.

DNA polymerase-β is required for 1-methyl-4-phenylpyridinium-induced apoptotic death in neurons

The biochemical pathways that mediate the degeneration of dopaminergic neurons in the substantia nigra of patients with Parkinson's disease are largely unknown. Recently, aberrant cell cycle events have been shown to be associated with neuronal death in several neurodegenerative diseases. In the present study, we investigated the role of DNA polymerases (DNA pols) in 1-methyl-4-phenylpyridinium (MPP(+))-induced neuronal apoptosis in cerebellar granule cells. After exposure to MPP(+), the neurons entered S phase of the cell cycle. Neuronal cell cycle re-entry and apoptosis were attenuated by flavopiridol, which is a broad inhibitor of cyclin-dependent kinases (CDKs). MPP(+) exposure significantly increased the expression of DNA pol-beta and primase but did not affect the expression of the canonical replicative DNA pols, including DNA pol-delta and pol-epsilon. Dideoxycytidine, which is a pharmacological inhibitor of DNA pol-beta, attenuated the neuronal apoptosis mediated by MPP(+). In a similar manner, the expression of a dominant negative form of DNA pol-beta was also neuroprotective. These results suggest that DNA pol-beta may have a causal role in MPP(+)-induced neuronal apoptosis.

Diverse and dynamic populations of cyanobacterial podoviruses in the Chesapeake Bay unveiled through DNA polymerase gene sequences

Many podoviruses have been isolated which infect marine picocyanobacteria, and they may play a potentially important role in regulating the biomass and population composition of picocyanobacteria. However, little is known about the diversity and population dynamics of autochthonous cyanopodoviruses in marine environments. Using a set of newly designed PCR primers which specifically amplify the DNA pol from cyanopodoviruses, a total of 221 DNA pol sequences were retrieved from eight Chesapeake Bay virioplankton communities collected at different times and locations. All DNA pol sequences clustered with the eight known podoviruses that infect different marine picocyanobacteria, and could be divided into at least 10 different subclusters (I-X). The presence of these cyanopodovirus genotypes based on PCR-amplification of DNA pol gene sequences was supported by the existence of similar DNA pol genotypes with metagenome libraries of Chesapeake Bay virioplankton assemblages. The composition of cyanopodoviruses in the Bay also exhibited distinct winter and summer patterns which were likely related to corresponding seasonal changes in the composition of cyanobacterial populations. Our study suggests that diverse and dynamic populations of cyanopodoviruses are present in the estuarine environment. The PCR method developed in this study provides a specific and sensitive tool to explore the abundance, distribution and phylogenetic diversity of cyanopodoviruses in aquatic environments. Linking the dynamics of host and viral populations in the natural environment is critical to broader characterization of the ecological role of virioplankton within microbial communities.

An 8-oxo-guanine repair pathway coordinated by MUTYH glycosylase and DNA polymerase λ

Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol lambda in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol lambda, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol lambda, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.

Structure and replication of yDNA: a novel genetic set widened by benzo-homologation.

In a functioning genetic system, the information-encoding molecule must form a regular self-complementary complex (for example, the base-paired double helix of DNA) and it must be able to encode information and pass it on to new generations. Here we study a benzo-widened DNA-like molecule (yDNA) as a candidate for an alternative genetic set, and we explicitly test these two structural and functional requirements. The solution structure of a 10 bp yDNA duplex is measured by using 2D-NMR methods for a simple sequence composed of T-yA/yA-T pairs. The data confirm an antiparallel, right-handed, hydrogen-bonded helix resembling B-DNA but with a wider diameter and enlarged base-pair size. In addition to this, the abilities of two different polymerase enzymes (Klenow fragment of DNA pol I (Kf) and the repair enzyme Dpo4) to synthesize and extend the yDNA pairs T-yA, A-yT, and G-yC are measured by steady-state kinetics studies. Not surprisingly, insertion of complementary bases opposite yDNA bases is inefficient due to the larger base-pair size. We find that correct pairing occurs in several cases by both enzymes, but that common and relatively efficient mispairing involving T-yT and T-yC pairs interferes with fully correct formation and extension of pairs by these polymerases. Interestingly, the data show that extension of the large pairs is considerably more efficient with the flexible repair enzyme (Dpo4) than with the more rigid Kf enzyme. The results shed light on the properties of yDNA as a candidate for an alternative genetic information-encoding molecule and as a tool for application in basic science and biomedicine.

Roles of specialized DNA polymerases in mutagenesis by oxidized guanine

To assess the involvement of specialized DNA polymerases in the mutagenesis induced by an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate), shuttle plasmid DNA containing the supF gene and 8-OH-dGTP were introduced into human 293T cells in which specialized DNA polymerases were knocked-down by siRNAs. The knock-downs of DNA polymerases eta and zeta, and REV1 reduced the A:T-->C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A. In contrast, the knock-down of DNA polymerase tau did not affect the mutations induced by 8-OH-dGTP. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols eta plus zeta and REV1 plus DNA pol zeta (but not by that of DNA pol eta plus REV1), suggesting that REV1-DNA pol eta and DNA pol zeta work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by 8-OH-dGTP.

RB69 DNA Polymerase Mutants with Expanded Nascent Base-Pair-Binding Pockets Are Highly Efficient but Have Reduced Base Selectivity

We have investigated the effect of systematically enlarging the nascent base-pair-binding pocket (NBP) of a replicative DNA polymerase from bacteriophage RB69 (RB69 pol) on the incorporation efficiency (k(pol)/K(d,app)) for both correct and incorrect dNMPs. Accordingly, we replaced residues L561, Y567, and S565 in the NBP with Ala, Ala, and Gly, respectively. We combined L561A and Y567A to give a double mutant and then introduced the S565G mutation to give a triple mutant. The efficiency of incorrect dNMP insertion increased markedly relative to the wild type with the single mutants and increased further as the number of substitutions in the NBP increased. The difference in incorporation efficiency for mispairs between the mutants and the wild-type RB69 pol was due mainly to k(pol). Unexpectedly, enlarging the NBP had a minimal effect on the incorporation efficiency of correct dNMPs. Our kinetic data suggest that replicative DNA pols exert base discrimination via "negative selection" against mispairs by using residues in the NBP, particularly the three residues analyzed in this study, to allow rapid incorporation of only correct base pairs. This proposal differs from how geometry and "tightness of fit" of the NBP is often invoked to account for rapid incorporation of correct base pairs, namely, that a tighter fit within the NBP leads to an increase in insertion rates [Kool, E. T. (2002) Annu. Rev. Biochem. 71, 191-219]. We related our findings to that of a model translesion DNA pol, Sulfolobus solfataricus Dpo4. We concur with the main conclusion of a previous study [Mizukami, S., et al. (2006) Biochemistry 45, 2772-2778], namely, that lesion bypass pols exhibit low incorporation efficiencies for correct dNMPs (leading to relative promiscuity) not because of a more open NBP but because of a loose fit of substrates bound in the catalytic centers. This is a property not shared by RB69 pol and its mutants.

Study of the inhibitory effect of fatty acids on the interaction between DNA and polymerase β

The binding of human DNA polymerase beta (pol beta) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol beta inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol beta inhibitors. In the interaction between pol beta and DNA, pol beta could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol beta inhibitors prevented the binding of pol beta to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol beta to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol beta-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol beta to DNA was mainly due to 7-fold increase in the dissociation rate.

Ctf4 coordinates the progression of helicase and DNA polymerase α

Ctf4 is a protein conserved in eukaryotes and a constituent of the replisome progression complex. It also plays a role in the establishment of sister chromatid cohesion. In our current study, we demonstrate that the replication checkpoint is activated in the absence of Ctf4, and that the interaction between the MCM helicase-go ichi ni san (GINS) complex and DNA polymerase alpha (Pol alpha)-primase is destabilized specifically in a ctf4Delta mutant. An in vitro interaction between GINS and DNA Pol alpha was also found to be mediated by Ctf4. The same interaction was not affected in the absence of the replication checkpoint mediators Tof1 or Mrc1. In ctf4Delta cells, DNA pol alpha became significantly unstable and was barely detectable at the replication forks in HU. In contrast, the quantities of helicase and DNA pol epsilon bound to replication forks were almost unchanged but their localizations were widely and abnormally dispersed in the mutant cells compared with wild type. These results lead us to propose that Ctf4 is a key connector between DNA helicase and Pol alpha and is required for the coordinated progression of the replisome.

The Block of DNA Polymerase δ Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase β and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol delta, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol beta and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase beta (pol beta), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol beta is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol beta or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol beta with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a "hit and run" mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

The Steric Gate Amino Acid Tyrosine 112 Is Required for Efficient Mismatched-Primer Extension by Human DNA Polymerase κ

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, which distinguishes dNTPs from rNTPs by sensing the 2'-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.

Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates: a convenient synthesis of useful probes for DNA polymerase beta structure and function.

Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase, and pyruvate kinase. DNA pol beta K(d) values for the alpha,beta-CF(2) and unmodified dNTPs, alpha,beta-NH dUTP, and the alpha,beta-CH(2) analogues of dATP and dGTP are discussed in relation to the conformations of alpha,beta-CF(2) dTTP versus alpha,beta-NH dUTP bound into the enzyme active site.

DNA polymerases with improved reverse transcriptase activity

There exists a need for enzymes with improved reverse transcriptase activity to increase the sensitivity of diagnostic assays which use RNA targets. In our lab we have produced a collection of chimeric mutant DNA polymerases with modulated proofreading activity. A mutagenized library of one of these DNA pols was screened for improved extension rates on a primed M13 template. Some of the mutants displayed the ability to perform improved RT‐PCR using either Mn2+ or Mg2+. One of the DNA pol mutations, D640G, was transferred to the parent DNA pols of the chimera, Thermatoga maritima and Thermus sp. Z05. These mutants also demonstrated the ability to perform efficient RT‐PCR with reduced RT incubation times.In Z05 the D580 position was mutated to all 19 other amino acids and the resulting proteins were purified. The D580G and K mutants were found to have improved RT activity and increased RT‐PCR sensitivity using TaqMan‐based detection. Enzymatic characterization revealed a 12.5‐fold improvement in the catalytic efficiency of Mn2+‐activated RT for Z05 D580G over wild‐type. The Z05 D580K enzyme also showed significant activity when Mn2+ was replaced by Mg2+ allowing for higher fidelity RT‐PCR.

DNA Synthesis across an Abasic Lesion by Human DNA Polymerase ι

Abasic sites are among the most abundant DNA lesions formed in human cells, and they present a strong block to replication. DNA polymerase iota (Poliota) is one of the few DNA Pols that does not follow the A-rule opposite an abasic site. We present here three structures of human Poliota in complex with DNAs containing an abasic lesion and dGTP, dTTP, or dATP as the incoming nucleotide. The structures reveal a mechanism of translesion synthesis across an abasic lesion that differs from that in other Pols. Both the abasic lesion and the incoming dNTPs are intrahelical and are closely apposed across a constricted active site cleft. The dNTPs partake in distinct networks of hydrogen bonds in the "void" opposite the lesion. These different patterns of hydrogen bonds, as well as stacking interactions, may underlie Poliota's small preference for insertion of dGTP over other nucleotides opposite this common lesion.

Comparative Efficacy of Conventional and TaqMan Polymerase Chain Reaction Assays in the Detection of Capripoxviruses from Clinical Samples

Sheeppox and goatpox are economically important viral diseases of sheep and goats, respectively. Both diseases are reportable to the World Organization for Animal Health. To implement a control and eradication program for these diseases, a rapid and user-friendly diagnostic tool is imperative for screening. Therefore, in the present study, TaqMan quantitative polymerase chain reaction (qPCR) and conventional PCR assays targeting the DNA polymerase (DNA pol) gene were developed for the detection of Capripoxvirus DNA from clinical specimens of sheep and goats. The 2 assays used different primer sets. Conventional PCR yielded a specific product of 134 bp, whereas qPCR yielded a 180-bp product. The specificity of amplified DNA pol gene products was confirmed by their size and by sequence analysis. The 2 assays were specific for Sheeppox virus and Goatpox virus. However, in comparison to conventional PCR, the qPCR was more rapid, specific, and 100 times more sensitive, with a detection limit as low as 0.042 pg of purified DNA. The qPCR assay was more sensitive (84.05%) than conventional PCR (76.06%) when used on clinical samples (n = 71) from sheep and goats.

A Polymerase Chain Reaction Strategy for the Diagnosis of Camelpox

Camelpox is a contagious viral skin disease that is mostly seen in young camels. The disease is caused by the Camelpox virus (CMLV). In the present study, a polymerase chain reaction (PCR) assay based on the C18L gene (encoding ankyrin repeat protein) and a duplex PCR based on the C18L and DNA polymerase (DNA pol) genes were developed. The former assay yields a specific amplicon of 243 bp of the C18L gene, whereas the duplex PCR yields 243- and 96-bp products of the C18L and DNA pol genes, respectively, in CMLV, and only a 96-bp product of the DNA pol gene in other orthopoxviruses. The limit of detection was as low as 0.4 ng of viral DNA. Both PCR assays were employed successfully for the direct detection and differentiation of CMLV from other orthopoxviruses, capripoxviruses, and parapoxviruses in both cell culture samples and clinical material. Furthermore, a highly sensitive SYBR Green dye-based, real-time PCR was optimized for quantitation of CMLV DNA. In the standard curve of the quantitative assay, the melting temperature of the specific amplicon at 77.6 degrees C with peak measured fluorescence in dissociation plot was observed with an efficiency of 102%. To the authors' knowledge, this is the first report to describe a C18L gene-based PCR for specific diagnosis of camelpox infection.

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

alpha-Accessory factor (AAF) stimulates the activity of DNA polymerase-alpha.primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44.AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-alpha.primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-(3)H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-alpha.primase template affinity and stimulate both DNA primase and polymerase-alpha activities in vitro.