Activating mutations in the NH2- and COOH-terminal moieties of the Gs alpha subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation.

The alpha subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The alpha s and alpha i2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH-terminal moiety of the alpha s polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH2-terminal half of the alpha s polypeptide encodes domains regulating beta gamma interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2- or COOH-terminal coding sequence of alpha s substituted with the corresponding alpha i2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the alpha s polypeptide. Mutation of either the amino- or carboxy-terminus results in an alpha s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated alpha s polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTP gamma S activation of adenylyl cyclase. In membrane preparations from cells expressing the various alpha s mutants, COOH-terminal mutants, but not NH2-terminal alpha s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTP gamma S and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the alpha s polypeptides. Thus, mutation at NH2- and COOH-termini influence the rate of alpha s activation, but only the COOH-terminus appears to be involved in the regulation of the alpha s polypeptide activation domain that interacts with adenylyl cyclase.     

A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity

The alphoid DNA-CENP-B (centromere protein B) complex is the first sequence-specific DNA/protein complex detected in the centromeric region of human chromosomes. In the reaction, CENP-B recognizes a 17-bp sequence (CENP-B box) and assembles two alphoid DNA molecules into a complex, which is designated complex A (Muro, Y., H. Masumoto, K. Yoda, N. Nozaki, M. Ohashi, and T. Okazaki. 1992. J. Cell Biol. 116:585-596). Since CENP-B gene is conserved in mammalian species and CENP-B boxes are found also in mouse centromere satellite DNA (minor satellite), this sequence-specific DNA-protein interaction may be important for some kind of common centromere function. In this study we have characterized the structure of CENP-B and CENP-B-alphoid DNA complex. We have shown by chemical cross-linking that CENP-B formed a dimer, and have estimated by molecular weight determination the composition of complex A to be a CENP-B dimer and two molecules of alphoid DNA. The DNA binding domain has been delimited within the NH2-terminal 125-amino acid region containing four potential alpha-helices using truncated CENP-B made in Escherichia coli cells. We have shown that CENP-B had sites highly sensitive to proteases and that the DNA binding domain was separable from the dimerizing activity by the proteolytic cleavage at 20 kD from the COOH terminus of the molecule. Thus, CENP-B may organize a higher order structure in the centromere by juxtaposing two CENP-B boxes in the alphoid DNA repeat through both the DNA-protein and protein-protein interactions.     

CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.     

Isolation of chromosome-associated proteins from Drosophila melanogaster that bind a human centromeric DNA sequence

The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is though to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.     

Crystal structure of the CENP-B protein-DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA.

The human centromere protein B (CENP-B), one of the centromere components, specifically binds a 17 bp sequence (the CENP-B box), which appears in every other alpha-satellite repeat. In the present study, the crystal structure of the complex of the DNA-binding region (129 residues) of CENP-B and the CENP-B box DNA has been determined at 2.5 A resolution. The DNA-binding region forms two helix-turn-helix domains, which are bound to adjacent major grooves of the DNA. The DNA is kinked at the two recognition helix contact sites, and the DNA region between the kinks is straight. Among the major groove protein-bound DNAs, this 'kink-straight-kink' bend contrasts with ordinary 'round bends' (gradual bending between two protein contact sites). The larger kink (43 degrees ) is induced by a novel mechanism, 'phosphate bridging by an arginine-rich helix': the recognition helix with an arginine cluster is inserted perpendicularly into the major groove and bridges the groove through direct interactions with the phosphate groups. The overall bending angle is 59 degrees, which may be important for the centromere-specific chromatin structure.     

Crystal structure of the human centromere protein B (CENP-B) dimerization domain at 1.65-A resolution

The human centromere protein B (CENP-B), a centromeric heterochromatin component, forms a homodimer that specifically binds to a distinct DNA sequence (the CENP-B box), which appears within every other alpha-satellite repeat. Previously, we determined the structure of the human CENP-B DNA-binding domain, CENP-B-(1-129), complexed with the CENP-B box DNA. In the present study, we determined the crystal structure of its dimerization domain (CENP-B-(540-599)), another functional domain of CENP-B, at 1.65-A resolution. CENP-B-(540-599) contains two alpha-helices, which are folded into an antiparallel configuration. The CENP-B-(540-599) dimer formed a symmetrical, antiparallel, four-helix bundle structure with a large hydrophobic patch in which 23 residues of one monomer form van der Waals contacts with the other monomer. In the CENP-B-(540-599) dimer, the N-terminal ends of CENP-B-(540-599) are oriented on opposite sides of the dimer. This CENP-B dimer configuration may be suitable for capturing two distant CENP-B boxes during centromeric heterochromatin formation.     

The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities.

Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.     

A new repetitive protein from Xenopus laevis skin highly homologous to pancreatic spasmolytic polypeptide

A cDNA sequence has been used to derive the precursor structure of a highly repetitive protein in Xenopus laevis skin. From the sequence of a whole family of secretory proteins can be predicted containing a classical hydrophobic signal sequence at the NH2-terminal end of the precursor. The proteins contain four domains with high homology to porcine pancreatic spasmolytic polypeptide. These four cysteine-rich, presumably physiologically active domains are separated in the molecule by a repetitive element, locating two such domains to the NH2 terminus of the precursor protein and the remaining two to the COOH-terminal end. The separating spacer consists of very unusual, precise, threonine and proline-rich repeats containing 9 residues which could be targets for extensive O-glycosylation. Additionally, processing at two pairs of basic residues is suggested to liberate two polypeptides ("spasmolysins") and "spasmolysin-glycoprotein."     

Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.     

A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay.

Human topoisomerase I is composed of four major domains: the highly charged NH(2)-terminal region, the conserved core domain, the positively charged linker domain, and the highly conserved COOH-terminal domain. Near complete enzyme activity can be reconstituted by combining recombinant polypeptides that approximate the core and COOH-terminal domains, although DNA binding is reduced somewhat for the reconstituted enzyme (Stewart, L., Ireton, G. C., and Champoux, J. J. (1997) J. Mol. Biol. 269, 355-372). A reconstituted enzyme comprising the core domain plus a COOH-terminal fragment containing the complete linker region exhibits the same biochemical properties as a reconstituted enzyme lacking the linker altogether, and thus detachment of the linker from the core domain renders the linker non-functional. The rate of religation by the reconstituted enzyme is increased relative to the forms of the enzyme containing the linker indicating that in the intact enzyme the linker slows religation. Relaxation of plasmid DNA by full-length human topoisomerase I or a 70-kDa form of the enzyme that is missing only the non-essential NH(2)-terminal domain (topo70) is inhibited approximately 16-fold by the anticancer compound, camptothecin, whereas the reconstituted enzyme is nearly resistant to the inhibitory effects of the drug despite similar affinities for the drug by the two forms of the enzyme. Based on these results and in light of the crystal structure of human topoisomerase I, we propose that the linker plays a role in hindering supercoil relaxation during the normal relaxation reaction and that camptothecin inhibition of DNA relaxation depends on a direct effect of the drug on DNA rotation that is also dependent on the linker.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein

Preparations of mannose-binding protein isolated from rat liver contain two distinct but homologous polypeptides. The complete primary structures of both of these polypeptides have been determined by sequencing of peptides derived from the proteins, isolation and sequencing of cDNAs for both proteins, and partial characterization of the gene for one of the proteins. Each polypeptide consists of three regions: (a) an NH2-terminal segment of 18-19 amino acids which is rich in cysteine and appears to be involved in the formation of interchain disulfide bonds which stabilize dimeric and trimeric forms of the protein, (b) a collagen-like domain consisting of 18-20 repeats of the sequence Gly-X-Y and containing 4-hydroxyproline residues in several of the Y positions, and (c) a COOH-terminal carbohydrate-binding domain of 148-150 amino acids. The sequences of the COOH-terminal domains are highly homologous to the sequence of the COOH-terminal carbohydrate-recognition portion of the chicken liver receptor for N-acetylglucosamine-terminated glycoproteins and the rat liver asialoglycoprotein receptor. Each protein is preceded by a cleaved, NH2-terminal signal sequence, consistent with the finding that this protein is found in serum as well as in the liver. The entire structure of the mannose-binding proteins is homologous to dog pulmonary surfactant apoprotein.     

Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen.

Proteolytic fragments of simian virus 40 tumor (T) antigen and T antigen that was dephosphorylated with alkaline phosphatase bound between 1.5 to 2 times more origin-containing simian virus 40 DNA than did intact T antigen in DNA saturation experiments. Kinetic experiments showed that these treatments also enhanced the rate at which T antigen bound to the DNA. The enhanced binding of T-antigen fragments correlated with the generation of DNA-binding fragments that lacked the NH2-terminal region. Dephosphorylation of T antigen in vitro resulted in the removal of phosphate groups from the NH2-terminal region as well as from the COOH-terminal region. To test the effects of dephosphorylation on the size of the protein, immunoaffinity-purified T antigen was subjected to sedimentation with and without prior treatment with alkaline phosphatase. Most of the purified protein sedimented as a monomer and no significant effect was observed after dephosphorylation, indicating that the enhanced DNA-binding activity was probably not due to the uncovering of additional binding sites buried specifically in oligomerized T antigen. Taken together, these results indicate that in vivo phosphorylation of the NH2-terminal region (residues 106 to 124) decreases the binding of the protein to the DNA origin. The effect is reversed by in vitro dephosphorylation or by proteolysis which removes the highly phosphorylated NH2-terminal arm of the polypeptide. We suggest that phosphorylation inactivates one of two distinct DNA-binding activities on the polypeptide chain perhaps corresponding to two separate regions in T antigen.     

Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain

A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.     

Extracellular release of colicin A is non-specific.

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.     

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.     

ATPase domain of eukaryotic DNA topoisomerase II. Inhibition of ATPase activity by the anti-cancer drug bisdioxopiperazine and ATP/ADP-induced dimerization

We have prepared full-length Drosophila and human topoisomerase II and truncation constructs containing the amino-terminal ATPase domain, and we have analyzed their biochemical properties. The ATPase activity of the truncation proteins, similar to that of the full-length proteins, is greatly stimulated by the presence of DNA. This activity of the truncation proteins is also sensitive to the inhibition by the drug bisdioxopiperazine, ICRF-193, albeit at a much lower level than the full-length protein. Therefore, bisdioxopiperazine can directly interact with the NH(2)-terminal ATPase domain, but the drug-enzyme interaction may involve other domains as well. The ATPase activity of the ATPase domain protein showed a quadratic dependence on enzyme concentration, suggesting that dimerization of the NH(2)-terminal domain is a rate-limiting step. Using both protein cross-linking and sedimentation equilibrium analysis, we showed that the ATPase domain exists as a monomer in the absence of cofactors but can readily dimerize in the presence of a nonhydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate. More interestingly, both ATP and ADP can also promote protein dimerization. This result thus suggests that the protein clamp, mediated through the dimerization of ATPase domain, remains closed after ATP hydrolysis and opens upon the dissociation of ADP.     

Interactions of colicin A domains with phospholipid monolayers and liposomes: relevance to the mechanism of action

The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592).     

Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain

Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.