Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

Abstract

African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3-5-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase ␤ (pol ␤), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol ␤-type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.Despite the variety of existing DNA polymerases, there are a few basic principles that are common to all these enzymes, irrespective of their role either in DNA replication or in DNA repair. The basic chemistry of each individual reaction always involves a pair of divalent metal ions that are coordinated by carboxylate residues. Such a two-metal ion mechanism, originally proposed by Beese and Steitz (1) and probably extrapolative to all nucleotidyltransferases (2-4), appears to be either evolutionarily conserved or acquired by convergent evolution of nonhomologous proteins. In addition to the general deoxynucleotidyl transfer mechanism, it appears that some structural convergency could apply also for the interaction with DNA, a common substrate; an overall view of the crystal structures available for DNA-dependent polymerases always shows a hand-shaped structure, with "thumb," "palm," and "fingers" subdomains, defining at least one cleft for holding DNA (5-12). Moreover, both DNA replicases and DNA repair enzymes are often multienzymatic proteins, having built-in nucleolytic activities that exist as individual structural modules, separated from the polymerization domain. Thus, the paradigmatic Escherichia coli DNA polymerase I (pol I) 1 has a proofreading 3Ј-5Ј-exonuclease, and a 5Ј-3Ј-exonuclease to remove the RNA from the Okazaki fragments (13). Most DNA replicases are also endowed with a proofreading 3Ј-5Ј-exonuclease domain, having an evolutionarily conserved pol I-type active site (14). Reverse transcriptases have an RNase H activity, required for second strand DNA synthesis (reviewed in Telesnitsky and Goff (15)). Even pol ␤, the smallest of the known DNA polymerases (39 kDa)...

Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

“…Homologs of all BER proteins except the oxidative DNA glycosylases have been identified and characterized in African Swine Fever Virus (AFSV) [46,47]. The giant Mimivirus also carries all BER proteins including a recently characterized DNA ligase [48] and the two oxidative DNA glycosylases, MvNei1 and MvNei2 described in this paper.…”

“…3) suggesting a role in the viral BER pathway. Homologs of human Polβ have been identified in both ASFV and Mimivirus [21,46]. The ASFV polX missing the N-terminal 8-kDa domain is unlikely to exhibit dRP lyase activity whereas the mimiviral PolX carries the putative dRP lyase domain.…”

Use of Damaged DNA and dNTP Substrates by the Error-Prone DNA Polymerase X from African Swine Fever Virus

“…Though data were not actually provided, Blanco and coworkers have indicated that Pol X is not expressed until the later stages of ASFV infection (52), whereas the ASFV replicative polymerase is expressed at both early and late stages of infection (59,60). It would therefore appear that if Pol X is indeed involved in TLS, it shows up at the last minute to rescue severely hindered replisomes.…”

“…Despite its relatiVely efficient usage of 8-oxo-dGTP, the minimal number of incorporation events that Pol X is expected to catalyze per genome [since it is a repair or perhaps a TLS, not a replicative, polymerase (9,51,52)] suggests that 8-oxo-G is probably not incorporated very frequently. Once incorporated into a single-nucleotide gap, persistence of 8-oxo-G in the genome would be dependent upon the relative efficiencies of the competing reactions catalyzed by ASFV APE (removal of 8-oxo-G via its 3′ f 5′ exonuclease editing activity) 8 and ASFV DNA ligase (sealing the 8-oxo-G-containing nick to give a stable, contiguous duplex).…”

Characterization of a Bacillus subtilis 64-kDa DNA Polymerase X Potentially Involved in DNA Repair

“…Additionally, the inhibition of the polymerization activity in the presence of ddNTPs is in agreement with a poor discrimination of the 3´-OH group of the incoming nucleotide, this fact being a general catalytic feature of this family of DNA polymerases 12,36,38 .…”