Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region.
Terminal deoxynucleotidyltransferase (Tdt) and DNA polymerase mu (pol mu) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol mu has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol mu with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol lambda does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol mu Loop1 reproduces pol mu behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called 'sequence determinant' regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition. These results are discussed in light of the available crystal structures.
Romain
Félix
F
Unité de Dynamique Structurale des Macromolécules and URA 2581 du C.N.R.S., Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
Barbosa
Isabelle
I
Gouge
Jérôme
J
Rougeon
François
F
Delarue
Marc
M
D000595:Amino Acid Sequence; D003035:Cobalt / Q000737:chemistry; D003461:Crystallography; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017384:Sequence Deletion; D013698:Templates, Genetic
D009711:Nucleotides; D003035:Cobalt; C415367:DNA polymerase beta2; C407743:DNA polymerase mu; D004253:DNA Nucleotidylexotransferase; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; C018021:cobaltous chloride