Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme.

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.

Gap-directed translesion DNA synthesis of an abasic site on circular DNA templates by a human replication complex.

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and ß are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, ß and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and ß, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, ß and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged "minicircle" DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and ß in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.

FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells.

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.

Mapping the functional yeast ABC transporter interactome.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.

The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability.

Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges. Together, our results suggest that BLM and RECQL4 have coordinated activities that promote genome stability.

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta.

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and ß are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, ß, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and ß, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol ß inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and ß are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and ß.

RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription.

It is known that transcription can induce DNA recombination, thus compromising genomic stability. RECQ5 DNA helicase promotes genomic stability by regulating homologous recombination. Recent studies have shown that RECQ5 forms a stable complex with RNA polymerase II (RNAPII) in human cells, but the cellular role of this association is not understood. Here, we provide evidence that RECQ5 specifically binds to the Ser2,5-phosphorylated C-terminal repeat domain (CTD) of the largest subunit of RNAPII, RPB1, by means of a Set2-Rpb1-interacting (SRI) motif located at the C-terminus of RECQ5. We also show that RECQ5 associates with RNAPII-transcribed genes in a manner dependent on the SRI motif. Notably, RECQ5 density on transcribed genes correlates with the density of Ser2-CTD phosphorylation, which is associated with the productive elongation phase of transcription. Furthermore, we show that RECQ5 negatively affects cell viability upon inhibition of spliceosome assembly, which can lead to the formation of mutagenic R-loop structures. These data indicate that RECQ5 binds to the elongating RNAPII complex and support the idea that RECQ5 plays a role in the maintenance of genomic stability during transcription.

Physical interaction of RECQ5 helicase with RAD51 facilitates its anti-recombinase activity.

Homologous recombination (HR) provides an efficient mechanism for error-free repair of DNA double-strand breaks (DSBs). However, HR can be also harmful as inappropriate or untimely HR events can give rise to lethal recombination intermediates and chromosome rearrangements. A critical step of HR is the formation of a RAD51 filament on single-stranded (ss)DNA, which mediates the invasion of a homologous DNA molecule. In mammalian cells, several DNA helicases have been implicated in the regulation of this process. RECQ5, a member of the RecQ family of DNA helicases, interacts physically with the RAD51 recombinase and disrupts RAD51 presynaptic filaments in a reaction dependent on ATP hydrolysis. Here, we have precisely mapped the RAD51-interacting domain of RECQ5 and generated mutants that fail to interact with RAD51. We show that although these mutants retain normal ATPase activity, they are impaired in their ability to displace RAD51 from ssDNA. Moreover, we show that ablation of RECQ5-RAD51 complex formation by a point mutation alleviates the inhibitory effect of RECQ5 on HR-mediated DSB repair. These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.

p300-mediated acetylation of the Rothmund-Thomson-syndrome gene product RECQL4 regulates its subcellular localization.

RECQL4 belongs to the conserved RecQ family of DNA helicases, members of which play important roles in the maintenance of genome stability in all organisms that have been examined. Although genetic alterations in the RECQL4 gene are reported to be associated with three autosomal recessive disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), the molecular role of RECQL4 still remains poorly understood. Here, we show that RECQL4 specifically interacts with the histone acetyltransferase p300 (also known as p300 HAT), both in vivo and in vitro, and that p300 acetylates one or more of the lysine residues at positions 376, 380, 382, 385 and 386 of RECQL4. Furthermore, we report that these five lysine residues lie within a short motif of 30 amino acids that is essential for the nuclear localization of RECQL4. Remarkably, the acetylation of RECQL4 by p300 in vivo leads to a significant shift of a proportion of RECQL4 protein from the nucleus to the cytoplasm. This accumulation of the acetylated RECQL4 is a result of its inability to be imported into the nucleus. Our results provide the first evidence of a post-translational modification of the RECQL4 protein, and suggest that acetylation of RECQL4 by p300 regulates the trafficking of RECQL4 between the nucleus and the cytoplasm.

MRE11 complex links RECQ5 helicase to sites of DNA damage.

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11-RAD50-NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3'-->5' exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.

The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase delta.

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL delta (hPOL delta). The hPOL delta enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL delta strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL delta. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL delta.

Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans.

BACKGROUND: Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of gamma-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. RESULTS: In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. CONCLUSION: Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the nucleotidyltransferase family. Characterization of another protein from the same operon revealed a 5'-polynucleotide kinase with a possible role in DNA strand-break repair.

Physical and functional interactions between Werner syndrome helicase and mismatch-repair initiation factors.

Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3'-5' exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSalpha), MSH2/MSH3 (MutSbeta) and MLH1/PMS2 (MutLalpha) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSalpha and MutSbeta can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3'-single-stranded arm. The stimulatory effect of MutSalpha on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutLalpha protein known to bind to the MutS alpha-heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutSalpha, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences.

DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance.

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda.

DNA polymerase lambda (pol lambda) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol lambda either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol lambda had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn++ and Mg++ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol lambda; (iii) pol lambda preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol lambda, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol lambda and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol lambda are discussed.

Phosphorylation of human DNA polymerase lambda by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen.

DNA polymerase (Pol) lambda is a member of the Pol X family and possesses four different enzymatic activities, being DNA polymerase, terminal transferase, deoxyribose phosphate lyase and polynucleotide synthetase, all localized in its C-terminal region. On the basis of its biochemical properties, Pol lambda has been implicated in various DNA repair pathways, such as abasic site translesion DNA synthesis, base excision repair and non-homologous end joining of double strand breaks. However, its role in vivo has not yet been elucidated. In addition, Pol lambda has been shown to interact with the replication clamp proliferating cell nuclear antigen (PCNA) in vitro and in vivo. In this work, we searched by affinity chromatography for novel partners and we identified the cyclin-dependent kinase Cdk2 as novel partner of Pol lambda. Pol lambda is phosphorylated in vitro by several Cdk/cyclin complexes, including Cdk2/cyclin A, in its proline-serine-rich domain. While the polymerase activity of Pol lambda was not affected by Cdk2/cyclin A phosphorylation, phosphorylation of Pol lambda was decreased by its interaction with PCNA. Finally, Pol lambda is also phosphorylated in vivo in human cells and this phosphorylation is modulated during the cell cycle.

Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases beta and lambda.

A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the alpha-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases alpha, beta and lambda, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase beta incorporated these derivatives in a strictly Mn++-dependent manner. On the other hand, polymerase lambda could incorporate some alkyltriphosphate derivatives with both Mg++ and Mn++, but only opposite to an abasic site on the template strand. The active site mutant polymerase lambda Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerases.

Diketo hexenoic acid derivatives are novel selective non-nucleoside inhibitors of mammalian terminal deoxynucleotidyl transferases, with potent cytotoxic effect against leukemic cells.

Mammalian terminal deoxyribonucleotidyl transferase (TDT) catalyzes the non-template-directed polymerization of deoxyribonucleoside triphosphates and has a key role in V(D)J recombination during lymphocyte and repertoire development. More than 90% of leukemic cells in acute lymphocytic leukemia and approximately 30% of leukemic cells in the chronic myelogenous leukemia crisis show elevated TDT activity. This finding is connected to a poor prognosis and response to chemotherapy and reduced survival time. On the other hand, recent data indicated that TDT is not the only terminal deoxyribonucleotidyl transferase in mammalian cells. Its close relative, DNA polymerase lambda, can synthesize DNA both in a template-dependent (polymerase) and template-independent (terminal deoxyribonucleotidyl transferase) fashion. DNA polymerase lambda might be involved in the nonhomologous end-joining recombinational repair pathway of DNA double-strand breaks. In this work, we report the characterization of the mechanism of action of three diketo hexenoic acid (DKHA) derivatives, which proved to be extremely selective for the terminal deoxyribonucleotidyl transferase activity of DNA polymerase lambda and TDT. They seem to be the first non-nucleoside-specific inhibitors of mammalian terminal transferases reported. Moreover, the DKHA analog 6-(1-phenylmethyl-1H-indol-3-yl)-2,4-dioxo-5-hexenoic acid (RDS2119) was not toxic toward HeLa cells (CC(50) > 100 muM), whereas it showed significant cytotoxicity against the TDT(+) leukemia cell line MOLT-4 (CC(50) = 14.9 muM), thus having the potential to be further developed as a novel antitumor agent.

Inhibition of mammalian DNA polymerases by resveratrol: mechanism and structural determinants.

Resveratrol, a natural compound found in many dietary plants and in red wine, plays an important role in the prevention of many human pathological processes, including inflammation, atherosclerosis and carcinogenesis. We have shown that the antiproliferative activity of resveratrol correlated with its ability to inhibit the replicative pols (DNA polymerases) alpha and delta in vitro [Stivala, Savio, Carafoli, Perucca, Bianchi, Maga, Forti, Pagnoni, Albini, Prosperi and Vannini (2001) J. Biol. Chem. 276, 22586-22594]. In this paper, we present the first detailed biochemical investigation on the mechanism of action of resveratrol towards mammalian pols. Our results suggest that specific structural determinants of the resveratrol molecule are responsible for selective inhibition of different mammalian pols, such as the family B pol alpha and the family X pol lambda. Moreover, the resveratrol derivative trans-3,5-dimethoxy-4-hydroxystilbene, which is endowed with a strong antiproliferative activity (Stivala et al., 2001), can inhibit pols alpha and lambda and also suppress the in vitro SV40 DNA replication. The potency of inhibition is similar to that of aphidicolin, an inhibitor of the three replicative pols alpha, delta and epsilon. Our findings establish the necessary background for the synthesis of resveratrol derivatives having more selective and potent antiproliferative activity.

Differential incorporation of halogenated deoxyuridines during UV-induced DNA repair synthesis in human cells.

Double labeling of interphase and metaphase chromosomes by 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) has been used in studies of the dynamics of DNA replication. Here, we have used this approach and confocal microscopy to analyze sites of DNA repair synthesis during nucleotide excision repair (NER) in quiescent human fibroblasts. Surprisingly, we have found that when both precursors are added at the same time to UV-irradiated cells they label different sites in the nucleus. In contrast, even very short periods of simultaneous IdU+CldU labeling of S-phase cells produced mostly overlapped IdU and CldU replication foci. The differential labeling of repair sites might be due to compartmentalization of I-dUTP and Cl-dUTP pools, or to differential utilization of these thymidine analogs by DNA polymerases delta and epsilon (Poldelta and Polepsilon). To explore the latter possibility we used purified mammalian polymerases to find that I-dUTP is efficiently utilized by both Poldelta and Polepsilon. However, we found that the UV-induced incorporation of IdU was more strongly stimulated by treatment of cells with hydroxyurea than was incorporation of CldU. This indicates that there may be distinct IdU and CldU-derived nucleotide pools differentially affected by inhibition of the ribonucleotide reductase pathway of dNTP synthesis and that is consistent with the view that differential incorporation of IdU and CldU during NER may be caused by compartmentalization of IdU- and CldU-derived nucleotide pools.