The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.

DNA polymerase (pol) ␦ is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol ␦ has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA͞DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA͞DNA primer, we could measure strand displacement by pol ␦ coupled to DNA synthesis. Our results suggested that pol ␦ alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol ␦, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20 -30 nucleotides, by generating a ''locked'' flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase ␦ is modulated by the concerted action of PCNA, RP-A and Fen 1.

The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols ␤ and and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol -dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol ␤ activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols and ␤. R eactive oxygen species (ROS) are produced during normal cell metabolism and through the action of exogenous agents (1). When ROS react with DNA, the most frequently generated lesion (10 3 to 10 4 per cell per day) is 7,8-dihydro-8-oxoguanine (8-oxo-G) (2), whose mutagenic potential in aging, tumor transformation, and neurodegenerative diseases is well established. The presence of 8-oxo-G in the replicating strand can lead to frequent misincorporation of A opposite the lesion by the human replicative DNA polymerases (pols) ␣, ␦, and (3). Full repair of 8-oxo-G lesion is guaranteed by 2 different base excision repair (BER) systems: (i) an OGG1-dependent, which targets C:8-oxo-G mispairs, removes the lesion and leaves an intact DNA strand to act as template for the resynthesis step (4); and (ii) a MUTYH-dependent pathway, which targets the A:8-oxo-G base pair and removes the adenine (5-7). Subsequent error-free bypass of the lesion requires a specialized DNA pol that can catalyze the correct incorporation of C opposite 8-oxo-G during the resynthesis step, reconstituting a C:8-oxo-G base pair that could subsequently be repaired by the OGG1-dependent BER. However, the majority of human DNA pols insert an adenine opposite 8-oxo-G on the template strand with high frequencies (10-75% of the time). Thus, the molecular mechanism ensuring correct and efficient repair of A:8-oxo-G mismatches in human cells is currently undetermined.We have recently shown (8) that the BER enzyme DNA pol , which belongs to DNA pol family X (9), is very efficient in performing error-free translesion synthesis past the 2 major oxidative lesions 8-oxo-G (8) and 2-hydroxy-adenine (2-OH-A) (10). Moreover, its fidelity and efficiency is enhanced 2 orders of magnitude by the auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A), both for normal and translesion synthesis, resulting in dATP incorporation frequencies opposite 8-oxo-G as low as 10 Ϫ3 . On the other hand, the other major BER enzyme DNA pol ␤ shows a relaxed nucleotide insertion specificity opposite 8-oxo-G, with erroneous (i.e., d...