A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication

DNA polymerases that initiate replication by protein-priming are able to catalyze terminal protein (TP)-primed initiation, the following transition steps and finally DNA-primed elongation. Therefore, their structures must be able to position sequentially both primers, TP and DNA, at a common binding site. For DNA-templated initiation, these DNA polymerases have to bind the origin of replication as template and TP as primer. It is likely that very precise interactions are required to position both TP and templating nucleotide at the polymerization active site. Such a specificity during TP-priming must rely on specific amino acids that must be evolutionarily conserved in this subfamily of DNA polymerases. By site-directed mutagenesis, we have analyzed the functional significance of Lys392 of phi29 DNA polymerase, immediately adjacent to the Kx3NSxYG motif, and specifically conserved among protein-primed DNA polymerases. During TP-primed initiation, mutations in this residue did not affect untemplated TP-dAMP formation, indicating that the interaction with the initiating nucleotide and TP were not affected, whereas the template-directed initiation activity was severely inhibited. Both mutant DNA polymerases had a wild-type-like (overall) DNA binding activity. We thus infer that residue Lys392 of phi29 DNA polymerase is important for the correct positioning of the templating nucleotide at the polymerization active site, a critical requirement during template-directed TP-priming at phi29 DNA origins. Consequently, mutation of this residue compromised the fidelity of the initiation reaction, not controlled by the 3'-5' exonuclease activity. During DNA-primed polymerization, the mutant polymerases showed a defect in translocation of the template strand. This translocation problem could be the consequence of a more general defect in the stabilization and positioning of a next templating nucleotide at the polymerization active site, during DNA-primed DNA synthesis.     

A positively charged residue of phi29 DNA polymerase, highly conserved in DNA polymerases from families A and B, is involved in binding the incoming nucleotide

Alignment of the protein sequence of DNA-dependent DNA polymerases has allowed the definition of a new motif, lying adjacent to motif B in the direction of the N-terminus and therefore named pre-motif B. Both motifs are located in the fingers subdomain, shown to rotate towards the active site to form a dNTP-binding pocket in several DNA polymerases in which a closed ternary complex pol:DNA:dNTP has been solved. The functional significance of pre-motif B has been studied by site-directed mutagenesis of 29 DNA polymerase. The affinity for nucleotides of 29 DNA polymerase mutant residues Ile364 and Lys371 was strongly affected in DNA- and terminal protein-primed reactions. Additionally, mutations in Ile364 affected the DNA-binding capacity of 29 DNA polymerase. The results suggest that Lys371 of 29 DNA polymerase, highly conserved among families A and B, interacts with the phosphate groups of the incoming nucleotide. On the other hand, the role of residue Ile364 seems to be structural, being important for both DNA and dNTP binding. Pre-motif B must therefore play an important role in binding the incoming nucleotide. Interestingly, the roles of Lys371 and Ile364 were also shown to be important in reactions without template, suggesting that 29 DNA polymerase can achieve the closed conformation in the absence of a DNA template.     

Two positively charged residues of phi29 DNA polymerase, conserved in protein-primed DNA polymerases, are involved in stabilisation of the incoming nucleotide

In DNA polymerases from families A and B in the closed conformation, several positively charged residues, located in pre-motif B and motif B, have been shown to interact with the phosphate groups of the incoming nucleotide at the polymerisation active site: the invariant Lys of motif B and the nearly invariant Lys of pre-motif B (family B) correspond to a His in family A DNA polymerases. In phi29 DNA polymerase, belonging to the family B DNA polymerases able to start replication by protein-priming, the corresponding residues, Lys383 and Lys371, have been shown to be dNTP-ligands. Since in several DNA polymerases a third residue has been involved in dNTP binding, we have addressed here the question if in the DNA polymerases of the protein-primed subfamily, and especially in phi29 DNA polymerase, there are more than these two residues involved in nucleotide binding. By site-directed mutagenesis in phi29 DNA polymerase the functional role of the remaining two conserved positively charged amino acid residues of pre-motif B and motif B (besides Lys371 and Lys383) has been studied. The results indicate that residue Lys379 of motif B is also involved in dNTP binding, possibly through interaction with the triphosphate moiety of the incoming nucleotide, since the affinity for nucleotides of mutant DNA polymerase K379T was reduced in DNA and TP-primed reactions. On the other hand, we propose that, when the terminal protein (TP) is present at the polymerisation active site, residue Lys366 of pre-motif B is involved in stabilising the incoming nucleotide in an appropriate position for efficient TP-deoxynucleotidylation. Although mutant DNA polymerase K366T showed a wild-type like phenotype in DNA-primed polymerisation in the presence of DNA as template, in TP-primed reactions as initiation and transition it was impaired, especially in the presence of the phi29 DBP, protein p6.     

Function of the C-terminus of phi29 DNA polymerase in DNA and terminal protein binding

The thumb subdomain, located in various family B DNA polymerases in the C-terminal region, has been shown in their crystal structures to move upon binding of DNA, changing its conformation to nearly completely wrap around the DNA. It has therefore been involved in DNA binding. In agreement with this, partial proteolysis studies of 29 DNA polymerase have shown that the accessibility of the cleavage sites located in their C-terminal region is reduced in the presence of DNA or terminal protein (TP), indicating that a conformational change occurs in this region upon substrate binding and suggesting that this region might be involved in DNA and TP binding. Therefore, we have studied the role of the C-terminus of 29 DNA polymerase by deletion of the last 13 residues of this enzyme. This fragment includes a previously defined region conserved in family B DNA polymerases. The resulting DNA polymerase Δ13 was strongly affected in DNA binding, resulting in a distributive replication activity. Additionally, the capacity of the truncated polymerase to interact with TP was strongly reduced and its initiation activity was very low. On the other hand, its nucleotide binding affinity and its fidelity were not affected. We propose that the C-terminal 13 amino acids of 29 DNA polymerase are involved in DNA binding and in a stable interaction with the initiator protein TP, playing an important role in the intrinsic processivity of this enzyme during polymerization.     

Loss of DNA polymerase beta stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity.

Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.     

Caught bending the A-rule: crystal structures of translesion DNA synthesis with a non-natural nucleotide

Damage to DNA involving excision of the nucleobase at the N-glycosidic bond forms abasic sites. If a nucleotide becomes incorporated opposite an unrepaired abasic site during DNA synthesis, most B family polymerases obey the A-rule and preferentially incorporate dAMP without instruction from the template. In addition to being potentially mutagenic, abasic sites provide strong blocks to DNA synthesis. A previous crystal structure of an exonuclease deficient variant of the replicative B family DNA polymerase from bacteriophage RB69 (RB69 gp43 exo-) illustrated these properties, showing that the polymerase failed to translocate the DNA following insertion of dAMP opposite an abasic site. We examine four new structures depicting several steps of translesion DNA synthesis by RB69 gp43 exo-, employing a non-natural purine triphosphate analogue, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP opposite abasic sites. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP. As with dAMP, the DNA fails to translocate following insertion of 5-NIMP, although distortions at the nascent primer terminus contribute less than previously thought in inducing the stall, given that 5-NIMP preserves relatively undistorted geometry at the insertion site following phosphoryl transfer. An open ternary configuration, novel in B family polymerases, reveals an initial template independent binding of 5-NITP adjacent to the active site of the open polymerase, suggesting that closure of the fingers domain shuttles the nucleotide to the active site while testing the substrate against the template.     

Interaction of DNA polymerase I (Klenow fragment) with the single-stranded template beyond the site of synthesis

Cocrystal structures of DNA polymerases from the Pol I (or A) family have provided only limited information about the location of the single-stranded template beyond the site of nucleotide incorporation, revealing contacts with the templating position and its immediate 5' neighbor. No structural information exists for template residues more remote from the polymerase active site. Using a competition binding assay, we have established that Klenow fragment contacts at least the first four unpaired template nucleotides, though the quantitative contribution of any single contact is relatively small. Photochemical cross-linking indicated that the first unpaired template base beyond the primer terminus is close to Y766, as expected, and the two following template bases are close to F771 on the surface of the fingers subdomain. We have constructed point mutations in the region of the fingers subdomain implicated by these experiments. Cocrystal structures of family A DNA polymerases predict contacts between the template strand and S769, F771, and R841, and our DNA binding assays provide evidence for the functional importance of these contacts. Overall, the data are most consistent with the template strand following a path over the fingers subdomain, close to the side chain of R836 and a neighboring cluster of positively charged residues.     

Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms

DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase–DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s⁻¹, much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.     

Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.

The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator.     

The properties of steric gate mutants reveal different constraints within the active sites of Y-family and A-family DNA polymerases

Y-family (lesion-bypass) DNA polymerases show the same overall structural features seen in other members of the polymerase superfamily, yet their active sites are more open, with fewer contacts to the DNA and nucleotide substrates. This raises the question of whether analogous active-site side chains play equivalent roles in the bypass polymerases and their classical DNA polymerase counterparts. In Klenow fragment, an A-family DNA polymerase, the steric gate side chain (Glu710) not only prevents ribonucleotide incorporation but also plays an important role in discrimination against purine-pyrimidine mispairs. In this work we show that the steric gate (Phe12) of the Y-family polymerase Dbh plays a very minor role in fidelity, despite its analogous role in sugar selection. Using ribonucleotide discrimination to report on the positioning of a mispaired dNTP, we found that the pyrimidine of a Pu-dPyTP nascent mispair occupies a similar position to that of a correctly paired dNTP in the Dbh active site, whereas in Klenow fragment the mispaired dNTP sits higher in the active site pocket. If purine-pyrimidine mispairs adopt the expected wobble geometry, the difference between the two polymerases can be attributed to the binding of the templating base, with the looser binding site of Dbh permitting a variety of template conformations with only minimal adjustment at the incoming dNTP. In Klenow fragment the templating base is more rigidly held, so that changes in base pair geometry would affect the dNTP position, allowing the Glu710 side chain to serve as a sensor of nascent mispairs.     

DNA Polymerase �� Substrate Specificity: SIDE CHAIN MODULATION OF THE ��A-RULE��*

Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) β, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol β insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol β bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.     

A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase.

Three conserved motifs (named A, B and C) have been proposed to form the polymerization active site in all classes of DNA-dependent polymerases. In eukaryotic-type (alpha-like) DNA polymerases, motif A is characterized by the consensus "Dx2SLYP". Mutants in phi29 DNA polymerase residue Tyr254 of this conserved motif had been previously shown to be affected in dNTP binding. Here, we show that a single substitution of Tyr254 into a valine residue enables the enzyme to incorporate ribonucleotide substrates, without affecting its wild-type affinity for dNTPs. Whereas the wild-type enzyme preferred dNTPs more than two million-fold over rNTPs, the mutation of Tyr254 into valine reduced the discrimination for rNTPs up to 1000-fold. In addition to this discrimination mechanism, based on sugar selection, phi29 DNA polymerase is very inefficient when extending an RNA primer terminus, allowing its exonucleolytic degradation. These results indicate that the Tyr254 of phi29 DNA polymerase is responsible for the discrimination against the 2'-OH group of an incoming ribonucleotide. This is the first time that the invariant tyrosine residue of motif A is involved in ribo- versus deoxyribonucleotide discrimination in an eukaryotic-type DNA polymerase.     

Characterization of a replicative DNA polymerase mutant with reduced fidelity and increased translesion synthesis capacity

Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase alpha, delta, epsilon or zeta, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure-function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 A crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol.     

An intramolecular FRET system monitors fingers subdomain opening in Klentaq1

A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA.     

Solution structure of a viral DNA repair polymerase.

DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP-bound conformation and high binding affinity for dGTP-Mg(2+) of Pol X may contribute to its low fidelity.     

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.     

Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7

The 2.25 A resolution crystal structure of a pol alpha family (family B) DNA polymerase from the hyperthermophilic marine archaeon Thermococcus sp. 9 degrees N-7 (9 degrees N-7 pol) provides new insight into the mechanism of pol alpha family polymerases that include essentially all of the eukaryotic replicative and viral DNA polymerases. The structure is folded into NH(2)- terminal, editing 3'-5' exonuclease, and polymerase domains that are topologically similar to the two other known pol alpha family structures (bacteriophage RB69 and the recently determined Thermococcus gorgonarius), but differ in their relative orientation and conformation.The 9 degrees N-7 polymerase domain structure is reminiscent of the "closed" conformation characteristic of ternary complexes of the pol I polymerase family obtained in the presence of their dNTP and DNA substrates. In the apo-9 degrees N-7 structure, this conformation appears to be stabilized by an ion pair. Thus far, the other apo-pol alpha structures that have been determined adopt open conformations. These results therefore suggest that the pol alpha polymerases undergo a series of conformational transitions during the catalytic cycle similar to those proposed for the pol I family. Furthermore, comparison of the orientations of the fingers and exonuclease (sub)domains relative to the palm subdomain that contains the pol active site suggests that the exonuclease domain and the fingers subdomain of the polymerase can move as a unit and may do so as part of the catalytic cycle. This provides a possible structural explanation for the interdependence of polymerization and editing exonuclease activities unique to pol alpha family polymerases.We suggest that the NH(2)-terminal domain of 9 degrees N-7 pol may be structurally related to an RNA-binding motif, which appears to be conserved among archaeal polymerases. The presence of such a putative RNA- binding domain suggests a mechanism for the observed autoregulation of bacteriophage T4 DNA polymerase synthesis by binding to its own mRNA. Furthermore, conservation of this domain could indicate that such regulation of pol expression may be a characteristic of archaea. Comparion of the 9 degrees N-7 pol structure to its mesostable homolog from bacteriophage RB69 suggests that thermostability is achieved by shortening loops, forming two disulfide bridges, and increasing electrostatic interactions at subdomain interfaces.     

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

Analysis of metal activation on the synthetic and degradative activities of phi 29 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 phi 29 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 phi 29 DNA polymerase. Furthermore, DNA competition experiments using phi 29 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 enzyme-DNA 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 phi 29 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.     

A conserved insertion in protein-primed DNA polymerases is involved in primer terminus stabilisation

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or alpha-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of phi29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3'-5' exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in phi29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.     

phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein

Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, phi29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.