Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α

Abstract

Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase ␣ (pol ␣). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol ␣ mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis but ϳ6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. cerevisiae DNA pol ␣. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases.During cell division, a mother cell divides to generate two identical daughter cells, each of which receives a complete genetic complement. DNA replication is a critical step, which duplicates the parental chromosomes prior to cell division in the cell cycle. In eukaryotic cells, at least three DNA polymerases participate in replication of nuclear DNA: DNA polymerases ␣ (pol ␣), 1 ␦, and ⑀, all of which belong to class B (pol ␣) DNA polymerases (1). The pol ␣/DNA primase complex plays a key role in the initiation phase of DNA replication; pol ␦ and pol ⑀ play roles primarily in the elongation phase. During initiation of DNA synthesis, DNA primase synthesizes a short RNA chain (ϳ10-mer), and pol ␣ extends the RNA primer by ϳ30 deoxyribonucleotides. Pol ⑀ and pol ␦ are highly processive DNA polymerases that carry out bidirectional DNA synthesis during the elongation phase of DNA replication (2).Class B family DNA polymerases share structures and catalytic mechanism with class A DNA polymerases such as Taq DNA pol I (3, 4). Motif B is an essential region conserved in class A DNA polymerases. A Lys residue is essential in motif B in Taq pol I (Lys-663) and Klenow fragment (Lys-758) of Escherichia coli pol I (5-7). Phe-667 in Taq (and the equivalent residues in Klenow and T7 pol) are also critical for polymerase activity, as well as discrimination of 2Ј,3Ј-dideoxyribonucleotides (5, 8, 9). Tyr-766 in Klenow fragment (Tyr-671 in Taq pol I) plays a role in fidelity of DNA synthesis (10, 11). Recently, Ponamarev et al. (12) reported that a human pol ␥ mutant in which a T...

The Gly-952 Residue of Saccharomyces cerevisiae DNA Polymerase α Is Important in Discriminating Correct Deoxyribonucleotides from Incorrect Ones

“…32 P-Labeled 15-mer (5Ј-CGC GCC GAA TTC CCN-3Ј), 16-mer (5Ј-CGC GCC GAA TTC CCG N-3Ј), 17-mer (5Ј-CGC GCC GAA TTC CCG CN-3Ј), or 18-mer (5Ј-CGC GCC GAA TTC CCG CTN-3Ј) primers were annealed to the 46-mer oligonucleotide, 5Ј-GCG CGG AAG CTT GGC TGC AGA ATA TTG CTA GCG GGA ATT CGG CGC G-3Ј, where N represents either one of the four nucleotides, and the underlined nucleotides were the target template sites. The reaction mixture (20 l) contained 100 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 , 50 mM KCl, 100 ng/l BSA, 2 mM DTT, 10 nM template-primer, various concentrations of dNTP, and an optimum concentration of each enzyme.…”

“…Site-directed Mutagenesis and Enzyme Preparation-Expression, purification, and specific activity determination of recombinant pol ␣ are described (32).…”

“…K D (DNA) values were determined with DNA concentration of 40 and 200 nM using the following equation, (38), in which k cat /K m , is an average of data from three independent experiments. Relative dissociation constant, K D (rel), was determined using the equilibrium binding method (32,38) as follows:…”

“…This motif in the P helix and residues in the N helix may together perform a comparable function to motif B (30,31). In the P helix of class B DNA polymerases, the distance between the Lys and Tyr is one residue shorter than in the O helix of class A DNA polymerases (32). As a result, the conserved residues in the putative ␣ helices do not have the same spatial relationships among class A and B polymerases and may be displaced by an approximately [1/4] helical turn.…”

“…As a result, the conserved residues in the putative ␣ helices do not have the same spatial relationships among class A and B polymerases and may be displaced by an approximately [1/4] helical turn. In the accompanying article (32), data are presented suggesting that conserved Gly-952 in Saccharomyces cerevisiae pol ␣ may play a similar role to motif B residue Tyr-951 in class A DNA polymerases. This idea is consistent with the hypothesis that the active sites of class A and B DNA polymerases are structurally distinct (32).…”