Metal activation of synthetic and degradative activities of .vphi.29 DNA polymerase, a model enzyme for protein-primed DNA replication

Abstract

Analysis of metal activation on the synthetic and degradative activities of 429 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3'+5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of 429 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'-5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and 429 DNA polymerase. Furthermore, DNA competition experiments using 429 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzymeDNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases, Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by 429 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP. I t has long been known that metal ions are required cofactors for the catalytic activities of DNA polymerases, Le., DNA polymerization and exonuclease activity (Bessman et al., 1958;Lehman & Richardson, 1964). Mg2+-activated catalysis has been the one most extensively studied, since, in general, it yields the highest activity. However, it has been demonstrated that other metal ions like MnZ+, Coz+, Ni2+, or Zn2+ can also serve as activators for DNA polymerases in vitro (Sirover & Loeb, 1976;Burgers & Eckstein, 1979), being presently unknown which metal or metals are used in vivo.Determination of metal ion requirements of DNA polymerases becomes complex by the fact that metals not only bind and activate DNA polymerases but also bind to DNA and dNTPs, leading to different template and substrate complexes depending on the metal ion and on its concentration (Murray & Flessel, 1976;Marzilli et al., 1980;Sigel, 1987). Furthermore, using a different approach, a single metal binding site has been detected in the presence of dGTP (Mullen et al., 1990). In the C-terminal domain of 429 DNA polymerase and other a-like DNA polymerases from distantly related organisms, a highly conserved amino acid sequence motif (YGDTDS) has been proposed to be part of the metal binding site for polymerization (Argos, 1988;Bernad et al., 1990). However, the role for this metal in binding dNTPs and primer substrates and/or catalysis is not yet clear.In the case of the 3' -5' exonuclease activity, a detailed understanding of the structural basis of the metal ion requirements has been provided by crystallographic analysis of pol I K complexed with ssDNA . The pol I K exonuclease active center contains two binding sites for metal ions: one met...

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme

“…In addition to earlier observations that DNA exonuclease domain of Phi29 DNA polymerase possesses DNA 39/59 exonucleolytic activity (Garmendia et al 1992;Esteban et al 1992), we show here that in the presence of divalent metal ions as cofactors the same enzyme exonucleolytically degrades ssRNA. The RNase activity acts in a 39 to 59 polarity.…”

Specific Recognition of Parental Terminal Protein by DNA Polymerase for Initiation of Protein-primed DNA Replication

“…The lack of activity of 29 pol⅐TP with TP-DNA templates from a different group of related phages, such as Nf (22) than Mg 2ϩ (Ref. 29 and this work), allowed detection of initiation using 29 pol⅐TP complexes with Nf templates. Initiation activity with both 29 and Nf TP-DNAs was measured using 29 and Nf initiation proteins.…”

“…The use of Mn 2ϩ instead of Mg 2ϩ in the in vitro system, which increases the formation of the initiation complex (Ref. 29 and this work), allowed us to detect initiation activity with pol⅐TP proteins from 29 or Nf and TP-DNAs from Nf or 29, respectively. This result prompted us to study the specificity of the template recognition by the initiation proteins (pol⅐TP).…”

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

“…We determined the concentration-dependent effects of Mg 2ϩ , Mn 2ϩ , and Ca 2ϩ on the translocation fluctuations, on primer strand transfer between the polymerase and exonuclease sites, and on dNTP binding, in individual ⌽29 DNAP-DNA complexes. Mg 2ϩ and Mn 2ϩ are metals that support catalysis in both the polymerase and exonuclease active sites, whereas Ca 2ϩ supports nucleotide binding but not catalysis in either the polymerase or the exonuclease active sites (34).…”