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A thorough molecular modeling study was carried out to explain the molecular basis of nonstructural protein 3 RNA helicase (NS3h) inhibition by QU663 which is one of a new generation of small-molecule nucleotide-mimicking inhibitors which are also potential anti-hepatitis C virus (HCV) agents. The resulting three-dimensional interaction model is discussed.

Research has been conducted on the NS3 ATPase/helicase. The interaction mechanism of the full-length NS3 ATPase/helicase with its nucleic acid and ATP substrate has been investigated and the results are reported.

DNA polymerase (pol) [delta] is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol [delta] has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA 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 RNA/DNA primer, we could measure strand displacement by pol [delta] coupled to DNA synthesis. Our results suggested that pol [delta] 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 [delta], 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 [delta] is modulated by the concerted action of PCNA, RP-A and Fen 1.

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 (ΔNS3) 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 ΔNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of ΔNS3 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 ΔNS3 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. [Copyright &y& Elsevier]

DNA pol (polymerase) is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol . RPA inhibited the elongation by human DNA pol on templates annealed to short primers. PCNA did not influence the elongation by DNA pol and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol to replicate past the AP site, independent of the primer length.

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.