title: Amino acid architecture that influences dNTP insertion efficiency in Y-family DNA polymerase V of E. coli.
abstract: Y-family DNA polymerases (DNAPs) are often required in cells to synthesize past DNA-containing lesions, such as [+ta]-B[a]P-N(2)-dG, which is the major adduct of the potent mutagen/carcinogen benzo[a]pyrene. The current model for the non-mutagenic pathway in Escherichia coli involves DNAP IV inserting deoxycytidine triphosphate opposite [+ta]-B[a]P-N(2)-dG and DNAP V doing the next step(s), extension. We are investigating what structural differences in these related Y-family DNAPs dictate their functional differences. X-ray structures of Y-family DNAPs reveal a number of interesting features in the vicinity of the active site, including (1) the "roof-amino acid" (roof-aa), which is the amino acid that lies above the nucleobase of the deoxynucleotide triphosphate (dNTP) and is expected to play a role in dNTP insertion efficiency, and (2) a cluster of three amino acids, including the roof-aa, which anchors the base of a loop, whose detailed structure dictates several important mechanistic functions. Since no X-ray structures existed for UmuC (the polymerase subunit of DNAP V) or DNAP IV, we previously built molecular models. Herein, we test the accuracy of our UmuC(V) model by investigating how amino acid replacement mutants affect lesion bypass efficiency. A ssM13 vector containing a single [+ta]-B[a]P-N(2)-dG is transformed into E. coli carrying mutations at I38, which is the roof-aa in our UmuC(V) model, and output progeny vector yield is monitored as a measure of the relative efficiency of the non-mutagenic pathway. Findings show that (1) the roof-aa is almost certainly I38, whose beta-carbon branching R-group is key for optimal activity, and (2) I38/A39/V29 form a hydrophobic cluster that anchors an important mechanistic loop, aa29-39. In addition, bypass efficiency is significantly lower both for the I38A mutation of the roof-aa and for the adjacent A39T mutation; however, the I38A/A39T double mutant is almost as active as wild-type UmuC(V), which probably reflects the following. Y-family DNAPs fall into several classes with respect to the [roof-aa/next amino acid]: one class has [isoleucine/alanine] and includes UmuC(V) and DNAP eta (from many species), while the second class has [alanine (or serine)/threonine] and includes DNAP IV, DNAP kappa (from many species), and Dpo4. Thus, the high activity of the I38A/A39T double mutant probably arises because UmuC(V) was converted from the V/eta class to the IV/kappa class with respect to the [roof-aa/next amino acid]. Structural and mechanistic aspects of these two classes of Y-family DNAPs are discussed.
authors.affiliation: Biology Department, Boston University, Boston, MA 02215, USA.
authors.forename: Kwang Young
authors.initials: KY
authors.lastname: Seo
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.forename: Prashant
authors.initials: P
authors.lastname: Donthamsetti
authors.forename: Sushil
authors.initials: S
authors.lastname: Chandani
authors.forename: Chui Hong
authors.initials: CH
authors.lastname: Lee
authors.forename: Edward L
authors.initials: EL
authors.lastname: Loechler
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D017104:Bacteriophage M13 / Q000235:genetics / Q000254:growth & development; D020134:Catalytic Domain; D004247:DNA; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D004247:DNA; D019951:DNA Polymerase beta; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase